University of Iowa

Iowa Research Online Theses and Dissertations

2008

Novel mechanisms for enzymatic regulation of phosphatidylcholine synthesis by proteolysis Beibei Chen University of Iowa

Copyright 2008 Beibei Chen This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/199 Recommended Citation Chen, Beibei. "Novel mechanisms for enzymatic regulation of phosphatidylcholine synthesis by proteolysis." PhD (Doctor of Philosophy) thesis, University of Iowa, 2008. http://ir.uiowa.edu/etd/199.

Follow this and additional works at: http://ir.uiowa.edu/etd Part of the Biochemistry Commons

NOVEL MECHANISMS FOR ENZYMATIC REGULATION OF PHOSPHATIDYLCHOLINE SYNTHESIS BY PROTEOLYSIS

by Beibei Chen

An Abstract Of a thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Biochemistry in the Graduate College of The University of Iowa

December 2008

Thesis Supervisor: Professor Rama K. Mallampalli

1 ABSTRACT Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Surfactant deficiency leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. These are features of the acute lung injury syndrome, characterized by a strong proinflammatory component where cytokines or bacteria infections greatly impair surfactant DPPtdCho biosynthesis. The key enzyme needed to produce surfactant DPPtdCho is a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCTα). Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilizes CCTα from actions of the proteinase, calpain. Mapping and site-directed mutagenesis of CCTα uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTα Q243 not only resulted in loss of CaM binding, but also led to complete calpain resistance in vitro and in vivo. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTα that stabilizes the enzyme under pro-inflammatory stress. We further show that CCTα does not undergo polyubiquitination and proteasomal degradation. Rather, the enzyme is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS resulting in disruption of its interaction with importin, nuclear exclusion, and subsequent degradation within the lysosome. Importantly, by using CCTαubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTα monoubiquitination masks its NLS resulting in cytoplasmic

2 retention.

These

results

unravel

a

unique

molecular

mechanism

whereby

monoubiquitination governs the trafficking of a critical regulatory enzyme in vivo. Last, we identify FBXL2 as a novel F-box E3 ubiquitin ligase that targets CCTα for degradation. Interestingly, FBXL2 also interacts with CaM, and CaM directly disrupts CCTα and FBXL2 interaction. This study demonstrates in the first time that adenoviral gene transfer of CaM attenuates the deleterious effects of P. aeruginosa infection by improving several parameters of pulmonary mechanics in animal models of sepsisinduced acute pulmonary injury. Collectively, these studies reveal a novel regulatory mechanism for phosphatidylcholine synthesis that may provide important clues to understanding the pathobiology of acute lung injury.

Abstract Approved: Thesis Supervisor Title and Department Date

NOVEL MECHANISMS FOR ENZYMATIC REGULATION OF PHOSPHATIDYLCHOLINE SYNTHESIS BY PROTEOLYSIS

by Beibei Chen

A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Biochemistry in the Graduate College of The University of Iowa

December 2008

Thesis Supervisor: Professor Rama K. Mallampalli

Graduate College The University of Iowa Iowa City, Iowa

CERTIFICATE OF APPROVAL

PH.D. THESIS This is to certify that the Ph.D. thesis of Beibei Chen has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Biochemistry at the December 2008 graduation.

Thesis Committee: Madeline A. Shea, Thesis Chair S. Ramaswamy Kris A. Demali Adrian Elcock Alexander Sandra

To my parents

ii

ACKNOWLEDGEMENTS This work would never have been accomplished without the assistance and encouragement of past and current members of the Mallampalli lab. In particular, I would like to thank my advisor, Rama Mallampalli for allowing me to grow as an independent thinker and scientist; Diann McCoy for her assistance on animal studies; Ron Salome for his assistance on LDH assays; Loree Henderson for her assistance on growing bacteria, Alan Ryan, Nancy Ray, Philip Butler for their support and advice during the writing of my manuscripts, grants and thesis. I am grateful to Madeline Shea for her guidance in this project. I also thank Chantal Allamargot and Alexander Sandra for their assistance on microscope imaging. I also thank Yalan for her assistance on mass spec analysis. This work was supported by an American Heart Association Predoctoral Fellowship Award (BBC), a Merit Review Award from the Department of Veteran's Affairs, and NIH R01 Grants HL081784, and HL080229 (to RKM).

iii

ABSTRACT Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Surfactant deficiency leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. These are features of the acute lung injury syndrome, characterized by a strong proinflammatory component where cytokines or bacteria infections greatly impair surfactant DPPtdCho biosynthesis. The key enzyme needed to produce surfactant DPPtdCho is a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCTα). Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilizes CCTα from actions of the proteinase, calpain. Mapping and site-directed mutagenesis of CCTα uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTα Q243 not only resulted in loss of CaM binding, but also led to complete calpain resistance in vitro and in vivo. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTα that stabilizes the enzyme under pro-inflammatory stress. We further show that CCTα does not undergo polyubiquitination and proteasomal degradation. Rather, the enzyme is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS resulting in disruption of its interaction with importin, nuclear exclusion, and subsequent degradation within the lysosome. Importantly, by using CCTαubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTα monoubiquitination masks its NLS resulting in cytoplasmic

iv

retention.

These

results

unravel

a

unique

molecular

mechanism

whereby

monoubiquitination governs the trafficking of a critical regulatory enzyme in vivo. Last, we identify FBXL2 as a novel F-box E3 ubiquitin ligase that targets CCTα for degradation. Interestingly, FBXL2 also interacts with CaM, and CaM directly disrupts CCTα and FBXL2 interaction. This study demonstrates in the first time that adenoviral gene transfer of CaM attenuates the deleterious effects of P. aeruginosa infection by improving several parameters of pulmonary mechanics in animal models of sepsisinduced acute pulmonary injury. Collectively, these studies reveal a novel regulatory mechanism for phosphatidylcholine synthesis that may provide important clues to understanding the pathobiology of acute lung injury.

v

TABLE OF CONTENTS LIST OF FIGURES………………………………….……………………………….…viii LIST OF ABBREVIATIONS………...………………………………………………..…xi CHAPTER I INTRODUCTION Eukaryotic PtdCho synthesis………………………………………………….......1 Calmodulin (CaM)………………………………………………………………...3 CCTα proteolysis by Calpain……………………………………………………...4 CCTα phosphorylation………………………………………………………….....5 Pseudomonas induced Lung Disease……………………………………………...6 CCTα protein degradation via ubiquitin-mediated processing…………..…….....7 Calcium and the lung…….………………………………………………………..8 Murine lung cell line………………………………………………………………9 Murine lung model…………..………………………………………………….....9 The focus of this thesis……………………………………..……………………10 CHAPTER II CALMODULIN BINDS AND STABILIZES THE REGULATORY ENZYME, CTP: PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE………...……….…………………..…..14 Abstract…………………………………………………………………………..14 Introduction………………………………………………………………………14 Materials and methods………………………………………………………….17 Results……………………………………………………………………………27 Discussion………………………………………………………………………..35 CHAPER III MASKING OF A NUCLEAR SIGNAL MOTIF BY MONOUBIQUITINATION LEADS TO MISLOCALIZATION AND DEGRADATION OF THE REGULATORY ENZYME, CCTα……..…58 Abstract……………………………………………………………………...….58 Introduction……………………………………….…………………………….58 Materials and methods…………………………….…………………………......61 Results………………………………………….…………………………...…..71 Discussion………..……………………………..………………………..……..83 CHAPTER IV THE NOVEL SCF FAMILY E3 LIGASE FBXL2 TARGETS CCTα FOR DEGRADATION…………….…………………………………125 Abstract…………………………………………………………………………125 Introduction……………………………………………………………………..125 Materials and methods………………………………………………………...127 Results………………………………………………………………………......135 Discussion………………………………………………………………………142 vi

CHAPTER V SUMMARY AND FUTURE STUDY……………………..…..…..…180 REFERENCES…………………………………………………………………………195

vii

LIST OF FIGURES Figure 1. Phosphatidylcholine synthesis pathway and domain structure of CCTα……...12 Figure 2. A PEST sequence is not required for calpain-mediated cleavage of CCTα…...40 Figure 3. CCTα is a CaM binding enzyme……………………………………………....42 Figure 4. CaM binds CCTα in vivo………………………………………………………44 Figure 5. Mapping of a CaM binding domain within CCTα…….………………………46 Figure 6. CaM binds within the CCTα membrane binding domain………...…………...48 Figure 7. Calmodulin protects CCTα from proteolysis………………………………….50 Figure 8. Endogenous CaM Stabilizes CCTα……………………………………………52 Figure 9. Mutagenesis of Q243 protects CCTα………….…………………………….54 Figure 10. CaM and calpain compete for CCTα binding via a CaM binding motif……56 Figure 11. CCTα is monoubiquitinated in vitro and in vivo……………………………..91 Figure 12. TNFα degradation of CCTα is mediated by the endosome-lysosomal pathway…………...…………………...…………………………….………..93 Figure 13. Apoptosis assays……………………………………………………………...95 Figure 14. Transfected CCTα localizes within nucleus………………………………….97 Figure 15. NH4Cl promotes CCTα translocation to cytosol……………………..……...99 Figure 16. TNFα accelerates CCTα translocation to cytosol…………………………...101 Figure 17. TNFα in combination with NH4Cl translocates CCTα to lysosome………..103 Figure 18. Ubi-CCTα fusion proteins localize in cytosol………………………………105 Figure 19. CCT-Ubiquitin fusion protein folds correctly………………………………107 Figure 20. Mapping of a ubiquitination site within CCTα……………………………..109 Figure 21. The K57R ubiquitin acceptor site within CCTα is functionally relevant…...111

viii

Figure 22. A K57R CCTα mutant exhibits greater protein stability and nuclear retention……………………………………..………………………….…..113 Figure 23. Monoubiquitination of CCTα K57 masks its nuclear localization signal…115 Figure 24. Monoubiquitination of CCTα at K57 disrupts its interaction with importin-α ………..……………………………………………………………………...117 Figure 25. Monoubiquitination of CCTα at K57 masks its nuclear localization signal and disrupts its interaction with importin-α……….…………..…………………119 Figure 26. Ubiquitin E3 machinery targets soluble CCTα……………………………..121 Figure 27. A model for CCTα nuclear activation, exclusion, and proteolytic processing mediated by protein monoubiquitination…………...……………………….123 Figure 28. Potential E3 ligase for CCTα………………………………………………..147 Figure 29. MALDI mass spec result……………………………………………………148 Figure 30. FBXO3 protein is identified………………………………………………..150 Figure 31. FBXO15 protein is identified…………………………………………….....152 Figure 32. FBXW4 protein is identified………………………………………………..154 Figure 33. FBXL2 protein is identified…………………………………………………156 Figure 34. FBXL2 E3 Ligase degrades CCTα………………………………………….158 Figure 35. Mapping of a CaM binding domain within CCTα…….……………………160 Figure 36. CaM and FBXL2 bind within the same motif of CCTα……………………162 Figure 37. FBXL2 interacts with N-terminal domain of CaM…………………………164 Figure 38. Conventional model and the proposed model for FBXL2 targeting CCTα...166 Figure 39. CaM competitively binds FBXL2 and disrupts the interaction between FBXL2 and CCTα………………………..………………………………………….168 Figure 40. Molecular model for FBXL2 targeting of CCTα …………………………..170 Figure 41. P. aeruginosa infection increases Ca2+ influx……………………………..172 Figure 42. Techniques used in flexiVent measuring lung mechanics……….………….174

ix

Figure 43. PA103 induced lung injury model…………………………………………..176 Figure 44. Replication-deficient adenovirus Calmodulin (Adv-CaM) gene transfer improves pulmonary function…………...………………………..…………178 Figure 45. Schematic diagram illustrating proposed pathways by which CCTα is regulated by CaM and Calpains…………………………………………….187 Figure 46. Schematic diagram illustrating proposed pathways by which CCTα is regulated by novel E3 ligase FBXL2……………………………………….189 Figure 47. Schematic diagram illustrating proposed pathways by which CCTα is regulated by FBXL2 and CaM…………………………………………...…191 Figure 48. Schematic diagram illustrating proposed pathways by which CCTα is regulated by deubiquitination enzymes (DUBs)……………………………193

x

LIST OF ABBREVIATIONS DPPtdCho ALI CCTα CK CPT CaM GST FBXL2 ATP BSA cDNA DMEM DNA DTT EDTA FBS HEPES kDa mRNA PAGE PBS PCR PMSF SDS siRNA PA103 ARDS CF SCF APC VCB UBC TNFα HCV NS5A

Dipalmitoylphosphatidylcholine Acute Lung Injury Choline-phosphate cytidylyltransferase A Choline kinase CDP phosphate transferase Calmodulin Glutathione-S-transferase F-BOX leucine rich protein 2 Adenosine triphosphate Bovine serum albumin Complementary DNA Dulbecco's modified Eagle's medium Deoxyribonucleic acid Dithiothreitol Ethylenediaminetetraacetic acid Fetal bovine serum N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) Kilo Dalton Messenger RNA Polyacrylamide gel electrophoresis Phosphate buffered saline Polymerase chain reaction Phenylmethylsulfonyl fluoride Sodium dodecyl sulfate Short interfering RNA Pseudomonas aeruginosa strand 103 Acute respiratory distress syndrome Cystic fibrosis Skp1-Cullin-F-box Anaphase-Promoting Complex VHL-Elongin C/Elongin B Ubiquitin conjugating enzyme (E2) Tumor Narcotic factor alpha Hepatitis C virus nonstructural protein 5A

xi

1 CHAPER I INTRODUCTION Pulmonary

surfactant

is

a

critical

substance

containing

dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins 1 that are produced and 2

The acute lung injury

syndrome (ALI) and chronic disorders such as cystic fibrosis

result in functional

secreted into the airspace from alveolar type II epithelial cells

surfactant deficiencies, leading to severe lung collapse, ventilatory difficulties, and gasexchange abnormalities that can lead to right heart failure. Clinical treatment of these disorders with surfactant replacement therapies is now underway

3,4

. However,

effectiveness of exogenous surfactant treatment in these disorders is limited because of the presence of infection with highly virulent strains of bacteria such as Pseudomonas aeruginosa (P. aeruginosa). Eukaryotic PtdCho synthesis Phosphatidylcholine (PC) accounts for ~60-70% of surfactant phospholipids 5. The major surface-active form of surfactant is dipalmitoylphosphatidylcholine (DPPtdCho). DPPtdCho biosynthesis in eukaryotic cells requires three steps as shown in Figure 1A : an ATP-dependent reaction in which choline is phosphorylated by choline kinase (CK), the conversion of cholinephosphate to CDP-choline by CTP:phosphocholine cytidylyltransferase (CCTα), and finally, the transfer of the choline phosphate moiety of CDP-choline to diacylglycerol to generate PtdCho by cholinephosphotransferase (CPT) 6. In this pathway, kinetic studies show that the formation of CDP-choline from cholinephosphate catalyzed by CCTα is very slow and rate-regulatory 7. There are four isoforms of CCT in cells: CCTα, CCTβ1, CCTβ2 and CCT β3. CCTα is the major

2 isoform in the lung and in surfactant-producing alveolar epithelial cells 8. The primary structure of CCTα has 367 amino acids and contains four functional domains, including an NH2-terminal nuclear localization signal, a catalytic core, an α-helical membrane binding domain, and a C-terminal phosphorylation domain (Figure 1B) 9. Because of its N-terminal nuclear localization signal, CCTα localizes exclusively in nucleus in all cell types except in lung epithelia cells. Endogenous CCTα is primary localized in the cytoplasm in MLE cells. Specifically, about 60~70% of CCT is localized in cytosol, whereas 30~40% of CCT is localized in nucleus by CCT immunostaining in MLE cells. Latest studies indicates that CCTα tend to relocalize to nucleus upon for example PGJ2 or calcium stimulation

10

. CCTα is an amphitrophic enzyme, thus can

switch between an inactive soluble or cytoplasmic form to an active, membrane-bound species within the nucleus. In fact, the ability of CCTα to reversibly translocate to nuclear envelops or endoplasmic membranes after stimulation by lipid activators is well established and remains central to enzyme activation 8. High CCT activities are detected in lung epithelia relative to other tissues

11-13

,

and pharmacological or genetic maneuvers directed at upregulating CCT activity are associated with increased surfactant PC levels; genetic defects in CCTα expression result in apoptosis and targeted CCTα gene deletion is embryonic lethal

8,14,15

. Therefore, CCT

has a distinct, regulatory role for the surfactant production making it an excellent target to regulate surfactant levels. The primary mechanisms by which CCTα activity is regulated include control by both activating and inhibitory lipids, by reversible phosphorylation, and by control at the level of mRNA 16. CCTα availability is also tightly controlled at the

3 level of enzyme turnover 8,17. Little is known about how these regulatory mechanisms are influenced by potential CCTα binding proteins. Calmodulin (CaM) CaM (16.7kD) is a calcium-binding protein that is highly conserved across species and exerts diverse biologic effects 18. There is very limited data, however, on the role of CaM as a regulator of lipid metabolism. Treatment with W7 calmodulin inhibitor, overcomes the decrease caused by calcitonin in serum lipids in rats, suggest that calcitonin

lowers

serum

lipid

levels

and

lipogenesis

in

hepatocytes

in

a

calcium/calmodulin-dependent way19. Genetically targeted expression of a CaM inhibitory peptide in surfactant-producing type II cells results in significantly delayed pulmonary development

20,21

. Inhibitors of CaM such as chlorpromazine and

trifluoperazine markedly increase phosphatidylserine synthesis, but decrease PC production 22. Further, lipid transfer protein nsLTP1 is regulated by CaM interaction in a calcium-independent manner 23. As a whole, these studies suggest that CaM might serve as a key modulator of CCT activity and surfactant lipid metabolism. CaM produces its physiologic effects via direct interaction with a broad range of proteins. Some proteins bind CaM in its calcium bound form (holoCaM), whereas others interact with CaM in a calcium-free form (apoCaM). CaM binding proteins are also classified into Ca2+dependent, Ca2+-independent, and Ca2+ inhibited proteins 24. Many CaM binding proteins are recognized by motifs characterized by a basic amphipathic helix, moderate to high helical hydrophobic moment, and a net positive charge 25. Other motifs described include an IQ motif (I/LQXXXRGXXXR), 1-8-14 CaM binding motif, and a 1-5-10 motif 26.

4 CCTα proteolysis by Calpain Prior studies in our laboratory indicate that CCTα is degraded, in part, by calcium-activated neutral proteinases (calpains)

17

. Calpains exist in cells as two major

isoforms depending on calcium requirements: M-calpain and μ-calpain. Each isoform consists of two distinct subunits, a larger 80-kDa catalytic subunit and a smaller 30-kDa regulatory subunit, forming a heterodimeric structure

27

. Calpains have diverse

physiological roles in the cells; they have been shown to be active participant in process such as cell mobility, cell cycle progression and apoptosis. Cellular calpains are able to be activated by a transient and localized influx of calcium, which then advance the signal transduction pathway by catalyzing the controlled proteolysis of its target proteins 28. For example, cerebrovascular accident or traumatic brain injury significantly increase in concentration of calcium in the cell results in calpain activation, which leads to random proteolysis of both target and non-target proteins and consequent irreversible tissue damage

29,30

. Calpains are well studied in brains, m-calpain is found in glia and a small

amount in axons, whereas μ-calpain is mainly located in the cell body and dendrites of neurons and to a lesser extent in axons and glial cells31. μ-calpain is determined to degrade cytoskeleton-associated paxillin, vinculin, talin, alpha-actinin and other cytoskeleton proteins, which regulates muscle growth and development

32,33

. However,

m-calpain which requires relatively high level of calcium (millimolar) to be activated, has shown to activates cellular apoptosis pathways

34

. Our lab has previous shown that μ-

calpain is a factor activated by injurious particle oxidized low density lipids (Ox-LDL) in lung

17

.

However, the other physiological roles of calpains in lung remain largely

unknown. Although structural properties direct calpains to their substrates, calpains also

5 recognize their substrates by two major motifs: PEST sequences and CaM-binding domains 35. There is also a CaM-like domain located within the small regulatory subunit of μ-calpain

27

. Of interest, calpain cleavage of IκBα involves binding of its PEST

domain with the CaM-like domain of calpain in a calcium dependent manner 35. Deletion of IκBα PEST domain abrogates degradation of IκBα by calpain. Also, association of IκBα with the “CaM-like” domain of calpain activates the catalytic domain of the proteinase, triggering calpain-mediated IκBα degradation. In fact, calcium-dependent binding of IκBα to calpain, but not catalysis, is the rate-limiting step in the cleavage reaction

35

. In this study, I investigated the hypothesis that CaM serves as a novel

binding partner for CCTα that modifies enzyme activity in the native state and in response to inflammatory events that trigger calpain-mediated CCTα degradation. CCTα phosphorylation CCTα activity is inhibited by phosphorylation in vitro and phosphorylation is restricted to sixteen serines within the carboxyl-terminal phosphorylation domain

36

.

There are also seven Ser followed by Pro suggestive of a role for proline-directed kinases such as p44 MAP kinase in CCTα control

37

. Indeed, our lab showed that p42/44 MAP

kinases inhibit PtdCho synthesis in vivo via site-specific docking and phosphorylation of CCTα

38

. CCTα phosphorylation influences membrane affinity, and membrane binding

and insertion are critical factors that stimulate CCTα function

39

. Membrane binding is

also associated with CCTα dephosphorylation, and phosphorylated CCT is less membrane associated 7. This may be attributed to electrostatic mechanisms, as CCTα phosphorylation imparts a negative surface charge on the enzyme thereby interfering with interactions between anionic phospholipids in the membrane and interfacial positively

6 charged lysines within the CCTα membrane binding domain

40

. It is known that CaM

modulates phosphorylation and membrane-association of proteins

41-44

. For example,

myosin light chain kinase (MLCK) phosphorylation of myosin is influenced by CaM 44. Pseudomonas induced lung disease P. aeruginosa is a well-recognized nosocomial and opportunistic pathogen that causes high morbidity and mortality and acute pulmonary exacerbations. This organism is the predominant isolate in ALI patients offending pathogen in cystic fibrosis

45

47

colonized with this pathogen by age 3

, in nosocomial pneumonia

46

, and the major

. For example 97% of children with CF are

48

. P. aeruginosa is a gram negative rod that

secretes multiple virulence factors, is genetically flexible, and triggers an exuberant host response 49. Major virulence factors include alginate, exotoxins A and exotoxins (Exo T, Exo S, Exo U, and Exo Y), that are injected from a type III secretory apparatus, quorumsensing for biofilm generation, and numerous other soluble products 49. P. aeruginosa has been shown to decrease surfactant levels in the baboon, rat, and mouse although the mechanisms for decreased lipids are not known

2,50-54

. Pseudomonas products also

degrade surfactant apoproteins involved in bacterial clearance

55-57

. P. aeruginosa

releases phospholipases that could potentially degrade surfactant DPPC releasing lysophosphatidylcholine, a hydrolysis product that causes lung injury

58-60

. In this

application, I tested the novel hypothesis that P. aeruginosa inhibits surfactant PC production by causing calcium influx, leading to degradation of CCTα by calpains and the SCF E3 ligase, FBXL2.

7 CCTα protein degradation via ubiquitin-mediated processing Protein ubiquitination has emerged as an important post-translational modification regulating lifespan of proteins in cells, but can also govern diverse processes such as gene transcription, endocytic vesicle trafficking, histone modification, and DNA repair

61,62

.

Conjugation of one (monoubiquitination) or multiple (multiubiquitination) ubiquitin molecules to lysines within target proteins serves as an important endocytic signal for internalization and targeting of various ion channels, membrane cargo receptors, and junctional proteins to the lysosomal/endocytic pathway 63. Polyubiquitination is a process by which a chain of at least four ubiquitin molecules are linked to a lysine on a substrate protein, most commonly resulting in degradation of the substrate protein via the 26S proteasome. Ubiquitination involves a series of steps: first, ubiquitin is activated in a twostep reaction by an E1 ubiquitin-activating enzyme, second, ubiquitin is transferred from E1-activating enzyme to an E2-ubiquitin conjugating enzyme, and third, an E3-ubiquitin ligase transfers ubiquitin from the E2 enzyme to a lysine residue on a substrate protein, resulting in an isopeptide bond between the substrate ε-amino lysine and the carboxylterminus of ubiquitin

64

. Ubiquitin ligation is the key step that confers substrate

specificity in this pathway, with hundreds of E3 ligases targeting specific substrates. There are at least four classes of E3 ligases: HECT-type, RING-type, PHD-type, and Ubox containing. There are also three major superfamilies of E3 ubiquitin ligase complexes: the SCF (Skp1-Cullin-F-box) family, the APC (Anaphase-Promoting Complex) family, and the VCB (VHL-Elongin C/Elongin B) family 65. Recently, the SCF family was shown to mediate targeting of transmembrane and membrane binding proteins for degradation

64

. The SCF complex has a catalytic core complex consisting of Skp1,

8 Cullin1 and the E2 ubiquitin-conjugating (Ubc) enzyme. The SCF complex also contains adaptor subunits, termed F-box proteins that target hundreds of substrates through specific domain interactions. F-box proteins have two domains: an NH2-terminal F-box motif and a carboxyl-terminal leucine-rich repeat (LRR) motif or WD repeat motif. The SCF complex uses the F-box motif to bind Skp1, whereas the leucine-rich/WD repeat motif is used for substrate recognition. F-box proteins have diverse roles including cell cycle regulation, synapse formation, plant hormone responses, and regulating the circadian clock. In this study, I showed that CCTα is degraded by an ubiquitin-regulated mechanism, an effect that appears to be directed by the SCF member, FBXL2. Also, I investigated for the first time the molecular basis for this interaction as a means to use CaM adenoviral gene transfer for alveolar epithelial expression in the setting of bacterial infection. Calcium and the lung Calcium ion plays an important role in surfactant ultrastructure and in surfactant secretion

66

. Although calcium concentrations in alveolar fluid are relatively high

(~2mM), lung epithelial cells maintain tight calcium homeostasis with very low intracellular calcium [Ca2+]i concentrations (nM-μM levels). Rises in [Ca2+]i are secondary to released Ca2+ from internal stores or by influx of extracellular Ca2+ through Ca2+ channels

62

. Intracellular Ca2+-binding proteins, such as calmodulin (CaM), act as

calcium sensors, decoding the information according to distinct increases in [Ca2+]I

67

.

Interestingly, in some clinical studies, calcium antagonists appear to improve pulmonary function in patients with ARDS

68

. Thus, we tested the hypothesis that, a P.

aeruginosa→calcium→ubiquitin E3 ligase→CCTα degradation pathway might be a

9 critical signaling mechanism by which P. aeruginosa exerts its suppressive effects on surfactant synthesis. Murine lung cell line Murine lung epithelial (MLE) cells are used extensively in the research of pulmonary surfactant production and regulation as well as lung development and tumorigenesis study, and it is the major cell line used in this thesis. This line is first established in 1992 by Kathryn A. Wikenheiser from pulmonary tumors in a mouse transgenic for the SV40 large T antigen under the control of the human surfactant protein C gene promoter 69. Specifically, lung tumors are collected from female transgenic mice within 4-5 months of age. The excised tumors and adjacent tissue are minced and washed with medium. Pooled washes and remaining tumor pieces are cultured separately in HITES medium, epithelial cells are isolated by colony selection 70. This cell lines exhibits rapid growth, lack of contact inhibition, and an epithelial cell morphology for 30-40 passages in culture. Microvilli, cytoplasmic multivesicular bodies, and multilamellar inclusion bodies (morphologic characteristics of alveolar type II cells) are detected by electron microscopic analysis

69

. The MLE cells also maintain functional characteristics

of distal respiratory type II epithelial cells including the expression of surfactant proteins B, C, and D and mRNAs. The cells secrete phospholipids in response to phorbol esters and ATP. Murine lung model Mice are frequently used to model respiratory diseases including asthma 71, ALI, 72

and pulmonary cystic fibrosis

73

. These diseases might result in alterations in the

respiratory rate, the tidal volume, the ratio of inspiratory to expiratory time, and airway

10 resistance and lung stiffness, that can be measured to model human diseases in murine systems

74

. The flexiVent is a unique, integrated platform for pre-clinical pulmonary

research. It has a fully computer-controlled architecture that generates widely used parameters such as dynamic Resistance, Compliance; Elastance and Pressure-Volume (PV). Resistance (R) assesses the level of constriction in the lungs. Compliance (C) captures the ease with which the lung can be extended. Elastance (E) captures the elastic rigidity of the lungs. Pressure-Volume (PV) loops capture the quasi-static mechanical properties of the respiratory system. In the P. aeruginosa induced murine ALI model, the stiffness of the lung results in higher resistance, lower compliance and higher elastance. In this study, I tested the novel hypothesis that overexpression of CaM by adenoviral gene transfer protects the murine lung in a P. aeruginosa induced lung injury model. The focus of this thesis The initial goal of this research was to determine how CCTα proteolysis is regulated by CaM in the native state or under pro-inflammatory conditions. For this purpose, an IQ-like CaM binding domain was discovered in CCTα; a CCTα Q243A mutant which is resistant to calpains was produced; in vivo and in vitro assays assessing CCT stability with CaM overexpression or knockdown were also executed. These results as shown in Chapter 2 are now published

21

. These observations also led to further

studies evaluating CCTα ubiquitination. I demonstrate that CCTα protein availability in cells is regulated by its rate of monoubiquitination, specifically at site K57; and monoubiquitinated CCTα translocates to lysosome from nucleus. These results as far were submitted to Nat. Struct. & Mol. Biol. and undergo final revision. Furthermore, a novel E3 ligase, FBXL2, was identified that targets CCTα. Interactions between FBXL2

11 and CaM was also examined to determine if these proteins are involved in forming a FBXL2/CCT/CaM complex. Other possible mechanisms for CaM regulation of CCTα are also discussed and tested. Last, in vivo animal studies confirmed that CaM protects CCTα and restores lung function.

12

Figure 1. Phosphatidylcholine synthesis pathway and domain structure of CCTα. A. (left panel) the major pathway in all mammalian cells for the production of DPPC is the Kennedy pathway (or CDP-choline pathway). This pathway involves three basic steps. The first step converts choline to phosphocholine, and this reaction is catalyzed by the enzyme choline kinase. The second reaction is a very slow reaction, which involves converting of phosphocholine to Cytidine-Diphosphocholine or CDPCholine. And this reaction is catalyzed by enzyme CCT or cytidylyltransferase. Last, CDP-Choline

is

then

coupled

to

Diaceyl-Glycerol,

resulting

the

product

phosphatidylcholine. And this reaction requires the enzyme CPT or Choline phosphotransferase. The phosphatidylcholine component is comprised predominantly or mostly of a molecule called DPPC or DI-Palmitate phosphatidylcholine, (right panel). And this molecule has Palmitate on the SN1 and SN2 position. B. The domain structure of CCTα. CCTα contains four functional domains, including a classical canonical nuclear localization signal, present in the first 40 residues; a mid portion catalytic core, a membrane binding domain which is important for enzyme activation, and last, a Cterminal phosphorylation domain; the role is yet to be determined.

13

14 CHAPTER II CALMODULIN BINDS AND STABILIZES THE REGULATORY ENZYME, CTP: PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE Abstract CTP: phosphocholine cytidylyltransferase (CCTα) is a proteolytically-sensitive enzyme essential for production of phosphatidylcholine, the major phospholipid of animal cell membranes. The molecular signals that govern CCTα protein stability are unknown. Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilized CCTα from actions of the proteinase, calpain. Adenoviral gene transfer of CaM in cells protected CCTα whereas CaM siRNA accentuated CCTα degradation by calpains. Mapping and site-directed mutagenesis of CCTα uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTα Q243 not only resulted in loss of CaM binding, but also led to　 complete calpain resistance in vitro and in vivo. Thus, calpains and CaM both access CCTα using a structurally similar molecular signature that profoundly affects CCTα levels. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTα that stabilizes the enzyme under pro-inflammatory stress. Introduction Tight homeostatic control of mammalian phosphatidylcholine (PtdCho) biosynthesis is critical to maintain cell membrane integrity and to ensure optimal secretion of key products, such as serum lipoproteins, bile, and pulmonary surfactant. In eukaryotes, the enzyme CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the pivotal step for PtdCho synthesis

75

. CCT is both rate-limiting and rate-regulatory for

15 PtdCho synthesis, and its deficiency is linked to apoptosis, impaired cell growth, and embryonic lethality 76. There are four isoforms of CCT in cells: CCTα, CCTβ1, CCTβ2 and CCTβ3

76,77

. The primary structure of the dominant species, CCTα, consists of 367

amino acids with four functional domains: an NH2-terminal nuclear localization signal (NLS), a catalytic core (C), a membrane-binding domain containing a hydrophobic ahelix (M), and a carboxyl-terminal phosphorylation domain (P). CCTα activity is regulated by activating and inhibitory lipids, by reversible phosphorylation, and at the level of mRNA 17. Little is known regarding other mechanisms that control its enzymatic behavior. CCTα protein turnover might be a physiologically important control mechanism as the enzyme is degraded by calcium-activated neutral proteinases (calpains) and death effector caspases 27. Calpain severs CCTα at the amino terminus and within the catalyticmembrane domain boundary

78,79

. Calpains exist in cells as two major isoforms

depending on calcium requirements: M-calpain and m-calpain. Each isoform consists of two distinct subunits, a larger 80-kDa catalytic subunit and a smaller 30-kDa regulatory subunit, forming a heterodimeric structure. The large and small subunits consist of four (I-IV) and two (I-II) domains, respectively 80. EF hand motifs within each subunit allow for heterodomain interactions and calcium binding. Calpain cleaves its substrates, in part, by docking to two major motifs within its substrates: PEST (proline glutamate serine threonine) sequences and calmodulin (CaM) binding domains. PEST sequences within IκBα and the ATP-binding cassette transporter 1 serve as proteolytic signatures for calpain degradation 81. Likewise, CaM binding domains within a calcium-ATPase pump and inducible nitric oxide synthase impact their sensitivity to calpain hydrolysis

82

. A

16 CaM domain also exists within the catalytic subunit of calpain that facilitates interaction with some PEST sequences or CaM binding motifs

83

. Indeed, database analysis

identified a consensus PEST sequence within the CCTα NH2-terminal domain

21

. Thus,

this PEST sequence or other structural motifs that recognize CaM might brand CCTα for its elimination within cells. Despite being a calpain substrate, CCTα is a relatively stabile enzyme. CCTα is highly abundant in cells, is cytosolic in pneumocytes, and has an extended half-life

84

.

Typically larger, hydrophobic, cytosolic proteins and regulatory enzymes exhibit faster turnover rates 40. CCTα’s half-life (~8 hrs) also exceeds that of other metabolic enzymes including HMG-CoA reductase and phosphoenolpyruvate carboxykinase (PEPCK)

83

.

These observations strongly suggest existence of covalent modifications, stabilizing ligands, or binding partners that enhance CCTα’s life-span in vivo. Calmodulin (CaM) (16.7kD) is a highly conserved calcium-sensing protein that binds and modulates stability of some cytoskeletal and ion transport proteins

83

, . CaM

binds proteins in its calcium bound (holoCaM) or calcium-free form (apoCaM). CaM binding proteins are thus classified into Ca2+-dependent, Ca2+-independent, and Ca2+ inhibited proteins. Many CaM binding proteins harbor recognition motifs characterized by a basic amphipathic helix, moderate to high helical hydrophobic moment, and a net positive charge. Other motifs described include an IQ motif (I/LQXXXRGXXXR), a 1-814, and 1-5-10 CaM binding motif. Although CaM protects some substrates from proteolysis, the molecular basis for these observations has not been fully elucidated. In the present study we investigated the hypothesis that specific molecular sequence signatures confer stability to CCTα. We show for the first time that CCTα is a

17 CaM-binding enzyme and that CaM protects CCTα from calpain degradation. A conceptually unique finding of this study is that a highly conserved residue (Q243) (rather than a NH2-terminal PEST sequence) serves as an essential molecular recognition site for competition between CaM and calpain for access to CCTα. Mutagenesis of Q243 within CCTα totally blocked CaM binding but also the ability of calpain to degrade CCTα in vitro and in vivo. The intermolecular competition between a proteinase and a stabilizing protein for access to a single recognition site within CCTα represents a novel mechanism regulating an enzyme’s availability. Materials and methods Materials Source of murine lung epithelial (MLE) cells, LDL, CCTα, ERK antibodies, TrueBlot IgG, FuGENE6 transfection kits, and TNT coupled reticulocyte lysate were described previously

38

. Rabbit polyclonal antibodies to M- and µ-calpain were from

ABR-Affinity BioReagents (Golden, CO). Rabbit monoclonal calmodulin antibody was purchased from Upstate (Billerica, MA). Rabbit polyclonal CaMKII antibody was purchased from Epitomics (Burlingame, CA). Purified µ-calpain recombinant CaM, and the calpain substrate peptide, LLVY, and trifluoperazine were purchased from Calbiochem (La Jolla, CA). The pCR-TOPO4 cloning kit, E. coli One Shot competent cells, pENTR Directional TOPO cloning kits, and the Gateway mammalian expression system were purchased from Invitrogen (Carlsbad, CA). The QuickChange site-directed mutagenesis kit and the X-blue cells were purchased from Stratagene (La Jolla, CA). The Gel extraction Kit and QIAprep Spin Miniprep Kit were from Qiagen (Valencia, CA). Nucleofector transfection kits were from Amaxa (Gaithersbury, MD). Calmodulin

18 Sepharose 4B beads were purchased from Amersham Biosciences (Sweden). Immobilized glutathione agarose beads were purchased from Pierce (Rockford, Illinois). BD TALON Purification and Buffer Kits were purchased from BD Biosciences (San Jose, CA). Calmodulin siRNA were purchased from Dharmacon (Chicago, IL). Cyclohexamide was from Biomol (Plymouth Meeting, PA). A mammalian calmodulin cDNA, pEx1-CaM, and was kindly provided by Dr. Madeline Shea (University of Iowa, Iowa City, IA)85. All DNA sequencing was performed by the University of Iowa DNA core facility. Construction of CCTα PEST mutants A CCTα variant harboring point mutations in the PEST domain (CCTPESTSDM) was constructed where Thr25 and Ser32 were mutated to Ala using the QuickChange site-directed mutagenesis kit. A full-length CCTα template (pCMV-CCTFL) plasmid DNA was used as a template. The primers used to mutate Thr25 were 5’GCCCTAATGGAGCAGCAGAGGAAGATGG-3’

(forward)

and

5’-

CCATCTTCCTCTGCTGCTCCATTAGGGC-3’ (reverse). The primers used to mutate Ser32 were 5’-GAAGATGGAATTCCTGCCAAAGTGCAGCGC-3’ (forward) and 5’GCGCTGCACTTTGGCAGGAATTCCATCTTC-3’ (reverse). PCR conditions were: initial denaturation at 95°C for 2 min, then denaturation at 95°C for 15s, annealing at 55°C for 30s, elongation at 68°C for 6min, 18 cycles for three steps. An internal deletion mutant lacking the PEST sequence (CCTPESTSOE) was constructed using splicing by overlapping extension PCR (SOE-PCR). Full-length CCTα cloned into TOPO4 (TOPO-CCTFL plasmid) was used as a template. Four primers CCTPEST1-4 were designed (CCTPEST1: 5’-CACCATGGATGCACAGAGTTCAGC3’,

19 CCTPEST2:

5’-

ACTGCACAGCGCTGCACTTTCCTCCTCTTCCTTGAATTGACTTTA-3’, CCTPEST3:

5’-

TAAAGTCAATTCAAGGAAGAGGAGGAAAGTGCAGCGCTGTGCAGT-3’

and

CCTPEST4: 5’-TCAGTCCTCTTCATCCTCGCTG-3’). In the first step, primers CCTPEST1 and CCTPEST2 were used to amplify an NH2-terminal fragment of CCTα. In the second step, primers CCTPEST3 and CCTPEST4 were used to amplify a Cterminal CCTα fragment. Each fragment flanked the PEST. In the last step, the two gel purified fragments from the steps above were used as a template in the final PCR reaction using primers CCTPEST1 and CCTPEST4 to amplify a desired 1050 bp product lacking the PEST. This fragment was purified and cloned into pPCR4-TOPO. The PCR conditions were as follows: 95 °C for 30 s and 18 cycles at 95 °C for 30 s, 55 °C for 60 s, and 68 °C for 5 min. After DNA sequence confirmation, TOPO-CCTPESTSOE was used as the template using the primers 5’- AAGCTTATGGATGCACAGAGTTCAGC-3’ (forward) and 5’-CTCGAGGTCCTCTTCATCCTCGCTG-3’ (reverse) to amplify the 1050 bp CCTα fragment. The forward primer contains a recognition site for HindIII; the reverse primer contains a recognition site for XhoI. The PCR product was cloned into pPCR4-TOPO followed by digestion with the same enzymes prior to directional cloning into pcDNA3.1/V5-his. Construction of GST-tagged CCTα domain and carboxyl-terminal mutants A series of internal CCTα domain deletion mutants were constructed using TOPO-CCTMEM and TOPO-CCTCAT that were first generated as described previously or by using SOE-PCR

38

. These constructs were used as a template in PCR to generate

20 GST-CCTMEM and GST-CCTCAT, two constructs devoid of the membrane binding domain

or

catalytic

domain,

respectively.

CACCATGGATGCACAGAGTTCAGCT-3’

The

and

forward

primer,

reverse

primer

5’5’-

GTCCTCTTCATCCTCGCTGA-3’ were used in the PCR conditions as follows: 95 °C for 30 s and 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min. The PCR products were gel purified and cloned into pENTR-TOPO. A series of carboxyl-terminal deletion mutants were constructed as follows: pCMV5-CCT was used as a template for PCR using the forward primer 5’CACCATGGATGCACAGAGTTCAGCT-3’ in combination with one of the following reverse

primers:

5’-ACTGATGGCCTGGAGCAT-3’

ACTTCCAATGAACTCTCGGG-3’

for

CACTTTCTGCACAAATTCTTTTGA-3’

TGACTTTTCCTCCACATCTTTCA-3’

for for for

CCT315,

CCT288,

5’5’-

CCT267,

5’-

CCT243,

5’-

for

TTGCAAGTGGTATTTCTTTTCGT-3’ GACAATGCGGGTGATGATGT-3’

for

CCT210, CCT260,

5’and

5’-

CCTTACCTTATCAACTCGTTCTTGC-3’ for CCT250. An NH2-terminal CCTα deletion mutant

(CCTND)

was

constructed

CACCTTACGGCAGCCAGCTCCT-3’

using and

the

forward

reverse

primer primer

5’5’-

GTCCTCTTCATCCTCGCTGA-3’. The PCR conditions were as follows: 95 °C for 30 s and 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min. All PCR products were gel purified and cloned into pENTR-TOPO. Finally, for cloning into GST fusion constructs, 150ng of pENTR-TOPO plasmids and 150 ng of pDEST27 GST destination vector were incubated with LR Clonase enzyme mix at 25°C for 1h per the

21 manufacturers’ instructions. The reactions were terminated by adding 1μl of proteinase K solution and heated at 37°C for 10 min. The plasmids were transformed into E. coli TOP10 competent cells, followed by plasmid preparation. A CCTα variant harboring a point mutation (CCTQ243A) where Gln243 was mutated to Ala was generated using the QuickChange site-directed mutagenesis kit. GSTCCTFL plasmid was used as a template for PCR using forward primer 5’GAAAAGAAATACCACTTGGCAGAACGAGTTGATAAGG-3’ and reverse primer 5’-CCTTATCAACTCGTTCTGCCAAGTGGTATTTCTTTTC-3’. The thermal cycling program was as follows: initial denaturation at 95°C for 2 min, then denaturation at 95°C for 30s, annealing at 55°C for 30s, elongation at 68°C for 10min, 18 cycles for three steps. The desired PCR product was gel purified and fused to GST as above. A CCTα carboxyl-terminal deletion mutant harboring a similar point mutation (CCT267Q243A) was generated by PCR using GST-CCT267 as the template using methods described above. All the above PCR products in pENTR-TOPO were verified by DNA sequencing. In vitro transcription and translation (TnT) CCTα cDNA constructs cloned into pCR4-TOPO4 (2mg plasmid/reaction) were added directly to the rabbit reticulocyte lysate, and incubated in a 50 ml of reaction solution containing 2 µg of plasmids, 25 µl rabbit reticulocyte lysate, 2.5 µl RNase Inhibitor, 1.2 µl of 1 mM amino acids (minus methionine), 2 µl T7 RNA Polymerase and 5 µl of [35S]-methionine (40µCi/reaction) were incubated at 30°C for 90 min as described 38

.

22 Calpain proteolysis assay A 25µl of reaction volume containing 10µl of TnT reaction products, 10µl calpain buffer (20mM Tris, pH7.5, 2mM dithiothreitol, 1% Tween 20, and 0.015% Triton X-100) and 0.25-1µg of purified µ-calpain was incubated at 37°C for 0-1h after adding CaCl2 to a final concentration of 200 µM. The reaction was terminated by adding 2X SDS protein loading buffer and heating to 95°C for 5 min. Effects of calpain hydrolysis of CCTα was further tested in separate studies by inclusion of varying concentrations of CCTα, CaM, or the calpain substrate, LLVY, in the reaction mixture. In these studies, calpain was present at a fixed concentration of 0.6 pmol/reaction. The digestion products were resolved by SDS-PAGE and gels processed for autoradiography or immunoblotting. In other experiments, purified recombinant GST- CCTα and GST- CCTα mutants (0.0050.05 mg) were used as substrates for calpain digestion. Cell culture MLE cells were cultured in Dulbecco's minimum essential medium (DMEM) containing 0% fetal bovine serum for up to 48h with or without Ox-LDL (100 µg/ml). Cells lysates were prepared by brief sonication in 150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM dithiothreitol, 0.025% sodium azide, and 1 mM phenylmethylsulfonyl fluoride (Buffer A) at 4 °C prior for analysis. Cytosolic and microsomal preparations were isolated as described 8. Lipoprotein oxidation Lipoproteins were dialyzed in phosphate-buffered saline at 4°C for 24h followed by oxidation in 5 µM CuSO4/PBS for 24h at 37 °C. Confirmation of lipoprotein

23 oxidization was by the malonaldehyde assay and by detection of apoprotein B-100 degradation as described 17. CCT activity Enzyme activity was determined by measuring the rate of incorporation of [methyl-14C] phosphocholine into CDP-choline using a charcoal extraction method

17

.

Assays were conducted with and without exogenous PtdCho/oleic acid lipid activator in the reaction mixtures. Immunoblot analysis Equal amounts of total protein in sample buffer were resolved using 10% SDSPAGE, transferred to nitrocellulose, and immunoreactive CCTα or calmodulin were detected as described

38

. Dilution factors for primary and secondary antibodies were

1:2000. CCTα was purified to homogeneity as described 86. Co-immunoprecipitations 200ug of total protein from MLE cell lysates were precleared with 20ul of Trueblot anti-Ig beads for 1h at 4 °C. 5µg of CCTα, ERK, CaM, rabbit IgG, or CaMKII antibodies were added for 2h incubation at 4°C. 20ul of Trueblot anti-Ig beads were added for additional 2h incubation. Beads were spun down and washed 5 times using 50 mM HEPES, 150 mM NaCl, 0.5 mM EGTA, 50 mM NaF, 10 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 1% (v/v) Triton X-100 (RIPA) buffer as described 38. Beads were heated at 100 °C for 5 min with 80µl of protein sample buffer prior to SDS-PAGE and immunoblotting.

24 Expression and knockdown of recombinant proteins CCTα PEST mutants were expressed in cells using the Amaxa nucleofector system per the manufacturers’ instructions. Cellular expression of green fluorescent tagged plasmids using this device was achieved at >90% in MLE cells. Transfection of GST-CCTα fusion constructs was conducted for 24h in DMEM/F12 medium containing 0% fetal bovine serum using 18μl of FuGene6 reagent and 6-10μg/dish of the desired plasmid. After 24h, the cells were harvested in Buffer A followed by brief sonication. In some studies, 24h after transfection, 100µg/ml of Ox-LDL was added for additional 24h. For overexpression of CaM, 1 x 106 cells were infected with Ad-CaM or an empty vector (Ad-Con) at MOI=40 for 6 h prior to an additional 24 h incubation with Ox-LDL. For CaM knockdown, 1 x 106 cells were electrophorated with 200 pmol of siRNA. Cells were either transfected with siRNA (sense sequence: 5’-UGACAAACCUUGGAGAGAAUU3’, antisense sequence: 5’-PUUCUCUCCAAGGUUUGUCAUU-3’) against CaM, or a control siRNA. 72 h after transfection, cells were exposed to Ox-LDL for an additional 24 h prior to harvest. In rescue studies, cells were either transfected with CaM siRNA or control siRNA; 48 h after transfection, cells were infected with Ad-CaM for another 24 h. Finally, Ox-LDL was added to the medium for an additional 24 h prior to harvest. CCTα degradation CCTα degradation was first determined by preincubating MLE cells for 24 h with cyclohexamide (0.5 mg/ml) followed by exposure of cells to DMEM medium alone or with trifluoperazine (30 mM) for various times prior to harvest for immunoblotting as above. In other studies, turnover of CCTα was determined by nucleofecting CaM siRNA or control siRNA as above, 72 h later cells were preincubated for 1 hr in methionine-

25 deficient medium and then pulsed with

[35]

S methionine (60 μCi/ml) for 4 h at 37°C as

described 8. Cells were rinsed, chased in medium replete with methionine and cysteine for 0 to 8 h, and processed for CCTα immunoprecipitation, SDS-PAGE, and autoradiography as described 17. GST pull down assays after plasmid transfection, cellular lysates were prepared as described above, followed by incubation with 20μl of immobilized glutathione agarose beads at 4°C for 1h. After incubation, the beads were spun down, rinsed 3 times using buffer containing 250mM NaCl and 0.2%NP-40. Beads were heated at 100 °C for 5 min with 80µl of protein sample buffer for subsequent immunoblotting. For purification of recombinant GST-CCTα, 100μl of immobilized glutathione agarose beads were incubated with cell lysates (prepared from two 100mm dishes) at 4°C for 4h. After incubation, beads were washed as described above. Recombinant GST- CCTα proteins were eluted using a 5mM glutathione Buffer A solution, followed by concentration using YM-30 spin columns. Calmodulin sepharose binding assay 20ul of CaM sepharose beads were incubated with cell lysates (30 mg) or purified rat liver CCTα (1-2 mg) with or without calcium at 4°C for 2h. After incubation, beads were spun down and washed 3 times using buffer containing 300mM NaCl and 0.1%NP40. Beads were heated at 100 °C for 5 min with 40µl of protein sample buffer. Released products were resolved using SDS-PAGE prior to immunoblotting. Mammalian two hybrid binding assay CCTα was PCR amplified using GST- CCTα as a template and cloned into the pM Vector (Clontech) that expresses the CCTα -Gal4BD fusion protein. CaM was also

26 PCR amplified using the Adv-CaM plasmid and cloned into a pVP16 Gal4AD Vector that expresses a CaM-Gal4AD fusion protein. After sequence confirmation, CCTαGal4BD, CaM-Gal4AD, and pG5CAT reporter vector were co-electrophorated into cells per the manufacturers’ instructions. 48 h after transfection, cells were lysed and assayed for b-galactosidase activities. pM-53 and pVP16-T plasmids were served as positive controls. pM3-VP16 and pVP16-CP plasmids were served as negative controls. Construction of an adenoviral-CaM vector A pEx1-CaM plasmid was used as a PCR template using the primers 5’CTCGAGATGGCTGACCAACTGACTGA-3’

(forward)

and

5’-

GGATCCCTTTGCTGTCATTTGTACAAAC-3’ (reverse) to amplify a 450 bp CaM fragment. The forward primer has an engineered XhoI site, and the reverse primer has an engineered BamHI site. PCR conditions were: 95°C for 30 sec and 35 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min. The PCR products were cloned into pPCR4-TOPO followed by digestion with XhoI and BamHI. The pacAD5 CMV IRES eGFP pA vector was also digested with XhoI and BamHI. These digestion products were fractionated by gel electrophoresis and the desired 450 bp and 7.5kb fragments were then purified and ligated using T4 DNA ligase at 37oC for 1h. The Gene Transfer Vector Core (University of Iowa) used the newly constructed adenovirus-CaM shuttle plasmid to generate a first generation adenovirus-CaM expression vector

87

.

Adenovirus vectors

expressing the CaM transgene driven by the CMV promoter (Adv-CaM) or an empty control vector containing the CMV promoter but no transgene (Adv-Con) were used in experiments. Adenoviral vectors were replication deficient (deletion of the E1 gene) and free of wild type contamination as determined and by plaque assay and by PCR for E1

27 sequences. The particle titers of adenoviral stocks were ~1012 particles/ml that were used in studies. Statistical analysis Statistical analysis was performed using one-way analysis of variance with a Bonferroni adjustment or a Student’s unpaired t-test. Data is expressed as m±SEM. Results CCTα PEST mutants are not resistant to Calpain A strong PEST sequence was identified in CCTα using the PEST-FIND algorithm (Fig. 2A). The calculated PEST score was +8.56, (PEST scores greater than +0 are considered highly significant). There are four less conserved CCTα PEST sequences, with scores ranging from -1.37 to -18.37. Mutagenesis was performed to disrupt this signature motif and potentially reduce ability of calpain to degrade CCTα. Mutagenesis of highly conserved Thr25 and Ser32 to Ala resulted in loss of the PEST sequence as identified by this algorithm. First, three constructs, CCTα full-length [CCTαFL]), and CCTSDM and CCTSOE (mutants with disrupted PEST motifs), were synthesized in vitro, incubated

with

calpain,

and

reaction

products

resolved

and

visualized

by

autoradiography. Calpain produced a dose-dependent decrease in the levels of the 42 kDa CCTαFL product (Fig. 2B, upper left). CCTαFL was degraded within 15 min of calpain exposure (Fig 2B, lower left). Calpain (0.5μg), effectively hydrolyzed CCTSDM and CCTSOE within 60 min (Fig. 2B, upper right panel) and at higher calpain concentrations (1 μg) levels of PEST mutants were undetectable within 30 min (Fig. 2B, lower right panel). Thus, disruption of the PEST sequence in CCTα does not protect against calpain in vitro. To assess the physiologic role of the CCTα PEST motif, functional CCTαFL or

28 CCTPESTSDM plasmids were transiently expressed in MLE cells prior to exposure to oxidized low-density lipoproteins (Ox-LDL) that activate calpains

17

. In MLE cells,

~60% of CCTα is detected in cytosol and the remainder membrane-associated (Fig. 2C). Ox-LDL reduced immunoreactive CCTα levels in both the cytosolic and membrane fractions. Ox-LDL uniformly produced a significant decrease in CCT activity to ~5060% of control values in untransfected cells when assayed in the absence of exogenous lipid (reflecting membrane-bound enzyme, Fig. 2D) and from 5.28±0.2 nmol/min/mg protein to 3.12±0.60 nmol/min/mg when assayed with exogenous lipid (total enzyme). Thus, Ox-LDL did not inactivate CCTα by simply shifting CCTα off the membrane into the cytosol, but by degrading the enzyme in both cellular compartments. Further, consistent with other calpain substrates, CCTα degradation products are not easily identified as these fragments are likely cleared rapidly in cells by the proteasome thereby often evading detection

78,88

. The sizes of products of the 20S and 26S proteasome are

only 500 kDa (5-6 residues) and would run near the dye front of the PAGE requiring more sensitive approaches for their detection. Oxidized lipids also impaired CCT activity in cells transfected with either CCTαFL or CCTPESTSDM (Fig. 2D). In these experiments, high-level expression of CCTαFL or CCTPESTSDM plasmids by nucleofection was observed; reductions of the levels of both overexpressed and endogenous CCTα proteins were observed after Ox-LDL treatment (Fig. 2E). These data indicate that the CCTα NH2-terminal PEST sequence within does not confer resistance to actions of calpains. CCTα is a Calmodulin (CaM) binding protein An alternative recognition signal for calpain in substrates is a CaM-binding domain

83

. We first investigated whether CCTα interacts with CaM. Thus, CaM or

29 extracellular

regulated

kinase

(ERK)

was

immunoprecipitated

followed

by

immunoblotting with anti-CCTα antibodies. CCTα was detected in association with immunoprecipitated CaM from cell lysates (Fig 3A [upper panel]); consistent with our prior studies, CCTα also bound to ERK, whereas this association was not detected using negative controls (rabbit IgG or beads alone). Conversely, immunoprecipitation of CCTα or CaMKII followed by immunoblotting with CaM antibody revealed that CaM was detected in association with CCTα and CaMKII (positive control), but not with preimmune serum (negative control) (Fig. 3A, [lower panel]). Next, cells were transfected with glutathione S—transferase- CCTα (GST- CCTα) fusion proteins and GST pull-down products were eluted and processed for CCTα and CaM immunoblotting (Fig. 3B). CaM was detected in association with overexpressed, purified GST- CCTα whereas this association was not demonstrated in untransfected cells or by using agarose beads. Further, cell lysates were run over CaM-agarose beads, the beads were extensively rinsed using buffer containing 0.1% NP-40, and products eluted. The elution products were then resolved by SDS-PAGE prior to CCTα immunoblotting. Different calcium concentrations ranging from 0 to 2000 mM were used in the binding buffer. Indeed, CaM interacts with CCTα in a calcium independent manner (Fig. 3C). To confirm a more direct interaction between CCTα and CaM, purified CCTα was run over CaM-agarose beads, and the beads were processed as above for CCTα binding (Fig. 3D). CaM was detected in association with incubation of 1-2 mg of purified CCTα. CCTα interacts with Calmodulin in vivo We tested CaM- CCTα binding using a mammalian two-hybrid assay (Fig. 4A). Cells were co-transfected with CCTα-Gal4BD and CaM-Gal4AD plasmids as fusion

30 proteins together with a plasmid construct encoding a b-galactosidase reporter gene (pG5CAT). CCTα -Gal4BD and CaM-Gal4AD transfected separately did not increase reporter activity (Fig. 4A, [inset]). Co-transfection of the CCTα -Gal4BD and CaMGal4AD plasmids together stimulated reporter activity comparable to the positive control plasmid indicative of CaM- CCTα binding. Together, these studies indicate that endogenous and overexpressed CCTα binds CaM in murine lung epithelia. Finally, we also employed FRET analysis using an acceptor photobleaching technique (Fig. 4B). In FRET, energy is transferred from a donor fluorophore molecule to an acceptor fluorophore molecule when proteins are in close (nanometer range) proximity. Thus, FRET is a powerful tool providing more direct visual evidence of protein-protein interaction in vivo. If FRET is observed using the acceptor photobleaching method, the donor emission (CFP) signal increases after a nearby acceptor fluorophore (YFP) is inactivated by irreversible photobleaching. Cellular transfection with YFP-CaM chimera led to diffuse cellular fluorescence of YFP-CaM in line with CaM localization in both cytosol and nucleus as described previously. Consistent with the ability of CaM to recruit binding partners to the nucleus

26

, co-transfection of these fluorescent plasmids led to

detection of a robust CFP-CCT signal within the nucleus, making CCT accessible to CaM (Fig. 4B, top). More importantly, the emission fluorescence values of both the donor CFP-CCT and acceptor YFP-CaM before and after acceptor photobleaching in the region of interest are shown (Fig. 4B, upper arrow and lower plots). These data show that upon bleaching, there was decreased acceptor fluorescence (YPF) coupled with an increase in donor emission fluorescence (CFP), because the acceptor cannot take in energy after its

31 photobleaching. As a whole, these data complement the physical interaction data in Fig. 3, demonstrating that CCT binds CaM in vivo and in vitro. Mapping the CaM binding site within CCTα CaM has a propensity to bind amphipathic a-helixes

89

helices within the CCTα membrane binding domain (Fig. 5A)

83

. There are two major . We hypothesized that

these helixes might harbor a potential CaM binding domain. We employed a reductionist approach by expressing GST-tagged CCTα constructs lacking functional domains (Fig. 5B). Following cellular plasmid transfection, lysates were resolved by SDS-PAGE followed by immunoblotting using GST antibodies to confirm expression of these mutants (Fig. 5C); cellular lysates were also run over glutathione-agarose beads, after stringent washing, GST pull-down products were eluted and resolved by SDS-PAGE prior to CCTα and CaM immunoblotting. As shown in Fig. 5C, each of these constructs was sufficiently expressed and exhibited appropriate mobilities as fusion proteins on immunoblots. CCTα immunoblotting as expected revealed a missing band after expression of GST-CCTCAT as the antisera is directed against the catalytic domain (Fig. 5D, [upper panel]). Full-length CCTα and GST- CCTα mutants devoid of the catalytic core (CCTCAT), PEST sequence (16-32) (CCTPEST), NH2-terminal sequence (1-40) (CCTN40), and carboxyl-terminus (315-367) (CCT315) all bound CaM (Fig. 5D, lower panel). However, deletion of the CCTα membrane binding domain (236-315) (CCTMEM) totally disrupted CaM- CCTα association. Thus, CaM binds CCTα within the membrane domain. We next tested several GST-CCTα mutants that were progressively truncated within the membrane-binding domain (at the carboxyl-terminus) to further localize a

32 CaM binding motif (Fig. 6A, upper map). C288 which retains helix1 and helix2 and C267 which contains helix1 both bound CaM (Fig. 6A, lower panel). However, binding of CaM was not observed with mutants C243 and C210. Thus, CaM binds CCTα in a span of residues from 243 and 267 in helix1. Additional mapping studies (Fig. 6B) revealed that C260 and C250 also bound CaM, thus localizing CaM interactions with CCTα to a motif, LQERVDKVK. This sequence displays some similarity to CaM IQbinding motifs with regard to conservation at Gln243 8. To evaluate the significance of this highly conserved site, we constructed a full-length CCTα and a truncated CCTα each with a single amino acid substitution at Q243 (FLQ243A and C267Q243). These mutants were tested in the GST pull-down interaction assay. The results (Fig. 6C) demonstrate that like the C243 construct, both FLQ243A and C267Q243 loose ability to interact with CaM. Thus, CCTα sequence 243LQERVDKD250 is required for CaM binding and Q243 is an important recognition site mediating this interaction. CaM modulates CCTα stability and function The above data suggest that CaM binds CCTα within a distinct recognition motif. As CaM stabilizes proteins, we next executed gain-of-function and loss-of-function analysis by manipulating its expression in vivo. First, purified CCTα was used in the calpain digestion assay in the absence or presence of exogenous CCTα, CaM, or the calpain peptide substrate, LLVY. Each reaction contained 0.6 pmole calpain. After hydrolysis, reaction products were processed for levels of immunoreactive CCTα and quantified by densitometry. As shown in Fig. 7A, 0.6 pmol of calpain effectively cleaved eighty percent of purified CCTα (0.7 pmol) in the absence of CaM; only at a ~ten-fold excess of recombinant CCTα (6 pmol) was calpain hydrolysis of the enzyme significantly

33 inhibited (Fig. 7A, left panel). Importantly, calpain-mediated CCTα hydrolysis was significantly reduced by either increasing the CaM/CCTα molar ratio or amounts of a calpain substrate in the reaction mixture (Fig. 7A, middle and right panels, respectively). In these studies, CCTα was present at 0.7 pmoles/reaction (Fig. 6A, middle and right panels). Of note, even at very low molar ratios (~0.5) of CaM/ CCTα, i.e. ~0.35 pmoles of CaM/0.7 pmoles CCTα, calpain hydrolysis of the substrate was inhibited by eighty percent (Fig. 7A, middle panel). Next, cells were infected with a replication-deficient adenovirus that expresses CaM (Adv-CaM) or an empty virus (Adv-Con) and 6 h later cells were exposed to Ox-LDL for 24 h. Adv-CaM, unlike the empty virus, totally blocked Ox-LDL inhibition of CCT activity (Fig. 7B) and CCTα degradation (Fig. 7C) while producing a robust increase in CaM levels (Fig. 7C). Next, MLE cells were either transfected with CaM siRNA or a control siRNA and 72 h later cells were exposed to OxLDL. As shown in Fig. 6D, Ox-LDL and CaM siRNA each significantly reduced CCT activity to ~ 40%-50% of control; CaM siRNA in combination with Ox-LDL accentuated this effect. These changes in enzymatic behavior were reflected in CCTα protein levels as CaM siRNA (while reducing CaM levels) significantly reduced steady-state CCTα mass, an effect accentuated when it was applied in combination with Ox-LDL (Fig. 7E). Finally, to assess a causal role for CaM in regulating CCTα availability, we conducted rescue experiments to determine if Ad-CaM can restore CCT function after CaM siRNA (Fig. 7F-G). Cells were either transfected with CaM siRNA or control siRNA; 48 h after transfection, cells were infected with Ad-CaM for 24 h and Ox-LDL was added to the medium for an additional 24 h. Ox-LDL inhibited CCT activity by ~30% in these studies, an effect that was enhanced in combination with CaM siRNA (Fig. 7F). Moreover, Ad-

34 CaM overexpression effectively restored CCT activity (Fig. 7F) and CCTα content (Fig. 7G) in cells after CaM siRNA transfection alone or in combination with Ox-LDL treatment. Together, these data indicate that CaM protects CCTα stability in vitro and in vivo. To confirm that endogenous CaM regulates CCTα protein stability, we performed CaM knockdown using siRNA, followed by measurements of CCTα half-life by pulsechase (Fig. 8, A and B). Consistent with prior studies, in the presence of scrambled siRNA, CCTα exhibited a t1⁄2 of ~8 h. CCT turnover was significantly accelerated in cells pretreated with CaM siRNA, since enzyme levels were almost undetectable by 2 h (Fig. 8B). These effects of CaM siRNA were associated with significant reductions in PtdCho synthesis (Fig. 8, C and D). As a whole, these observations indicate that endogenous CaM stabilizes CCTα in vivo. A Q243A mutant confers resistance to Calpain Calpain efficiently cleaves its substrates, in part, by docking to CaM binding motifs within target substrates. As Q243A totally disrupts CaM binding, we investigated whether this site was also critical for calpain recognition. GST-CCTFL and GSTCCTQ243A were purified and used as substrates for calpain digestion. While GST-CCTFL was efficiently cleaved by calpain resulting in appearance of several hydrolysis products in a dose dependent manner, the GST-CCTQ243A mutant was totally resistant to calpain cleavage (Fig. 9A). These results were confirmed with higher doses of calpain (0.5 mg) where the GST-CCTQ243A mutant was still resistant to calpain cleavage. Cells were transfected with GST-CCTQ243A to assess its physiologic role in response to Ox-LDL. Ox-LDL decreased the activity of both endogenous and overexpressed full-length CCTα

35 to approximately 66% of control values. However, Ox-LDL did not significantly decrease CCT activity in cells expressing GST-CCTQ243A (Fig. 9B). Immunoblotting experiments revealed that both endogenous CCTα and GST-CCTFL were degraded by Ox-LDL whereas levels of the 68-kDa GST-CCTQ243A fusion product displayed considerable stability comparable to untreated controls. Thus, Gln243 may serve as a key dock site for competitive access of CaM and calpain within the CCTα enzyme and its mutagenesis significantly blocks proteinase activity in vitro and in vivo. Discussion To the best of our knowledge, these studies demonstrate a novel regulatory model whereby competition between a proteinase and CaM for access to a single, highly conserved residue (Q243) within a CaM binding motif profoundly affects levels of an enzyme. This specific site (Q243) in CCTα appears to serve as a structurally unique recognition signal for both calpains and CaM that may vie for occupancy within CCTα. This site, rather than a consensus PEST sequence, is integrally linked to CCTα proteolysis by calpains. The data also represent the first demonstration that CCTα is a CaM binding protein and that CaM antagonizes CCTα degradation by calpains in vitro and in vivo. Evidence supporting CaM interactions with CCTα include: (i) coimmunoprecipitation of CCTα with CaM from cellular lysates, (ii) immunodetection of CaM in association with GST- CCTα fusion proteins in GST pull-downs, (iii) detection of CCTα in CaM-sepharose binding assays, and iv) mammalian two-hybrid analysis. Manipulation of CaM expression by adenoviral overexpression or CaM siRNA in lung epithelia differentially altered stability of CCTα in the native state and in response to destabilizing effects of oxidized lipoproteins. Thus, CaM appears to be a physiologically

36 relevant binding partner for CCTα that may serve to protect the enzyme from proteolytic cleavage. Although the ratio of immunoreactive CaM versus CCTα levels in lung epithelia appear relatively low, this does not preclude CaM as a bona fide binding partner that protects CCTα under native conditions from calpains. The relative amounts of CaM, calpains, and CCTα in cells depend, in part, upon the avidity of antibodies for their detection. CCTα stability will also be governed by the binding affinities of calpains versus CaM for CCTα. Thus, high affinity binding of CaM to CCTα might be sufficient to overcome lower stoichiometries of CaM relative to other proteins. CCTα is proteolytically sensitive to calpains in vitro, but yet exhibits a relatively long half-life in cells suggesting the presence of a stabilizing binding partner for the enzyme. The extended half-life of CCTα is somewhat unexpected as calpains are constitutively active in cells that would shorten the enzyme’s lifespan. Our data showing that CaM siRNA accelerate CCTα degradation in cells provides strong evidence that endogenous CaM is sufficient to protect CCTα from degradation from calpain (Fig. 7). Thus, the relatively slower turnover rate of CCTα compared to other metabolic enzymes could be explained by CaM-bound CCTα that masks recognition sites for calcium-activated proteinases under both native conditions and in settings of proteinase excess. Calpains, in part, target PEST motifs within substrates to facilitate degradation 90. The program PESTFIND identified a strong PEST motif (residues 16-32) within CCTα evidenced by a hydrophilic stretch of amino acids containing two prolines, several acidic residues, and one serine flanked by an NH2-terminal lysine. Mutagenesis of threonine25 and serine32 to alanine within CCTα removed this motif as a high value PEST target.

37 Indeed, both the CCTα T25A/S32A double mutant and an internal deletion mutant devoid of the PEST motif retained sensitivity to calpain degradation in vitro and after cellular expression. As with c-fos and Ca++-ATPase, the CCTα PEST motif may not serve as a proteolytic signal 91 with the caveat that the PEST domain is sufficiently exposed in vivo. Unlike cyclic AMP dependent kinase

83

, the PEST motif is probably unmasked in our

system because ~fourty-percent of CCTα was membrane-associated, a feature that activates CCTα exposing its NH2-terminal domain 84. A CaM binding motif within CCTα might serve as an alternative recognition signal for calpains. Database analysis of the CCTα sequence initially predicted a putative CaM binding domain within the distal catalytic-membrane domain interface (residues 205-240) on the basis of hydrophobicity, an average hydrophobic moment, and propensity for a-helix formation. Mapping studies using GST-CCTα carboxyl-terminal truncated mutants however localized a CaM binding motif to residues 242-250 exclusively within the membrane-binding domain (Fig. 6). This motif has some features resembling other CaM binding domains. This region resides within α-helix-1, consistent with the predilection of CaM binding domains to localize in amphipathic helices

83

.

Second, the presence of a calpain cut site juxtaposed upstream of this domain is in line with calpain cleavage of substrates at or near CaM binding domains

83

(Fig. 4A).

However, the sequence, LQERVDKVKKKVK, is not identical to, yet exhibits some similarity to IQ motifs (IQXXXRGXXXR) present in proteins that bind CaM in a calcium-independent manner 92. The CaM binding domain within CCTα harbors a highly conserved glutamine at position 2 and a basic residue at position 11, features characteristic of IQ motifs 93.

38 A remarkable observation from our studies is that a single amino acid substitution of glutamine within the CaM binding motif (Q243A) not only negates CaM binding, but was sufficient to totally block degradation of CCTα by calpain (Fig. 7A). These effects were recapitulated in cells exposed to oxidized lipids, where the expressed Q243A CCTα mutant construct was resilient to calpains evidenced by stability of the overexpressed protein and preservation of enzyme activity. We did not examine the functionality of other residues as Glu is highly conserved within the CaM motif. Presumably, polarity and/or electrostatic interactions between Q243 or other residues in the motif and calpain might enhance accessibility of the proteinase to its adjacent CCTα cleavage site or help sequester calcium (Fig. 10). Of note, Gln within the motif may also have several favorable electrostatic interactions with the backbone of CaM at Leu111, Gly112 and Glu113 or via binding to domain IV of the large calpain subunit at basic loops (1AJI.pdb) Our results differ from studies of the calcium ATPase and inducible nitric oxide synthase where deletion of an entire canonical CaM binding region either attenuated or was insufficient to modify calpain activity 94. Conversely, removal of CaM binding domains within caldesmon and calponin do not alter substrate recognition by calpain. Unlike our results, Padanyi et. al. demonstrated that point mutagenesis at Trp1093 in the calcium ATPase pump increased accessibility of a calpain hydrolysis site within a CaM binding domain . Thus, our data suggest a somewhat unique molecular model whereby availability of CCTα will be influenced by stoichiometry and binding affinities of CaM versus calpain utilizing Q243 as a critical recognition site. Interestingly, a point mutation at a highly conserved Gln (Q3180P) was recently identified within an IQ motif of the gene encoding abnormal spindle-like microcephaly associated (ASPM) protein; this

39 mutation is linked to an inherited disorder characterized by neurodevelopmental arrest of brain growth raising the possibility that interactions between ASPM, calpains, and CaM might play a role in disease pathogenesis . Our study demonstrates that CaM stabilizes a protein in cells, an issue not addressed in prior work. Of note, CaM inhibition reduces PtdCho synthesis and impairs lung growth, but these studies relied on use of nonselective approaches. Adenoviral gene transfer of CaM into lung epithelia was achieved with high efficiency (>95%) and specificity allowing for gain-of-function analysis. Complementary loss-of-function experiments were facilitated by cellular electroporation of CaM siRNA constructs that also proved to be efficient (Fig. 7). Adv-CaM overexpression effectively blocked OxLDL-induced CCTα breakdown and rescued acceleration of enzyme turnover after CaM knockdown indicating that siRNA effects were selective for CaM. An important observation these studies coupled with our results of physical protein interaction data underscore a potentially important biochemical and physiologic role for CaM in regulating PtdCho biosynthesis. Future work using structural analysis of CaM- CCTα complexes will provide newer insight into the conformational environment for these interactions. Testing of these mechanistic associations in vivo also await generation of suitable transgenic or knock-in animal model systems that conditionally express relevant molecular sites within CCTα or CaM in epithelia.

40

Figure 2. A PEST sequence is not required for Calpain-mediated cleavage of CCTα.

A. The black box identifies a highly conserved PEST sequence within CCTα. The dashed-underlined sequences are weak PEST sequences. The amino acids pointed by black arrows were mutated to alanine to disrupt the PEST sequence. B. Full-length CCTα (CCTFL), or CCTα variants with PEST mutations (CCTSDM) or an internal PEST deletion mutant (CCTSOE) were translated in vitro using a rabbit reticulocyte system. Newly synthesized products were incubated with calpain at various concentrations (upper) or times (lower) in a proteolysis assay. After hydrolysis, products were run on SDS-PAGE and gels dried prior to autoradiography. C. Murine lung epithelial (MLE) cells were cultured for 24 h with or without Ox-LDL (100μg/ml) prior to harvest and immunoblotting for CCTα and b-actin levels within microsomal and cytosolic fractions. D. MLE cells were nontransfected (NT) or transfected with CCTFL or CCTPESTSDM. After 24 h, Ox-LDL (100μg/ml) was added for an additional 24 h and cells were harvested and analyzed for CCT activity (D) or immunoblot analysis (E). Results are mean ± SEM from n=3 experiments (panel C, n=#2).

41

42

Figure 3. CCTα is a CaM binding enzyme

A. Coimmunioprecipitation. MLE cells were lysed and incubated with TrueBlot beads alone, or with rabbit IgG, calmodulin (CaM), or ERK1 polyclonal antibodies (upper panel). Cells were also lysed and incubated with beads alone, or with rabbit IgG, CCTα, or calmodulin kinase II (CaMKII) polyclonal antibodies (lower panel). Immunoprecipitants were resolved by SDS-PAGE prior to CCTα (upper panel) or CaM (lower panel) immunoblotting. CCTα migrates at ~42kDa whereas CaM　 is detected at ~17 kDa; the band (lower panel) at ~23 kDa is Ig light chain. B. GST-pull downs. A GST- CCTα fusion construct (10μg plasmid/dish) was transfected in cells using FuGene6. After transfection, cell lysates were incubated with glutathione-agarose beads, rinsed, and products eluted and resolved by SDS-PAGE prior to CaM immunoblotting. Nontransfected (NT) cell lysates or agarose beads alone processed similarly served as a negative control. C-D. CaM-sepharose binding assay. Cell lysates (30 mg) (C) were incubated with CaM-sepharose beads at different Ca++ concentrations. After extensive rinsing, elusion products were resolved by SDS-PAGE prior to CCTα immunoblotting. Cell lysates were incubated with agarose beads as a negative control. D. Purified CCTα (1-2 μg) was incubated with CaM-Sepharose beads and processed as above for CCTα immunoblotting.

43

44

Figure 4. CaM binds CCTα in vivo

A. mammalian two-hybrid assay. Cells were co-transfected using electroporation with CCT-Gal4BD (CCTα) and CaM-Gal4AD (CaM) plasmids as fusion proteins separately (inset) or in combination with a plasmid construct encoding a β-galactosidase reporter gene (pG5CAT) per the manufacturers’ instructions. Cells were lysed and assayed for β-galactosidase activities. pM-53/pVP16-T and pM3-VP16/pVP16-CP plasmids served as positive and negative controls, respectively. p G(1) transition. Journal of Biological Chemistry 274, 26240-8 (1999).

109.

Carter, R.S., Pennington, K.N., Arrate, P., Oltz, E.M. & Ballard, D.W. Sitespecific monoubiquitination of IkappaB kinase IKKbeta regulates its phosphorylation and persistent activation. J Biol Chem 280, 43272-9 (2005).

110.

Ea, C.K., Deng, L., Xia, Z.P., Pineda, G. & Chen, Z.J. Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol Cell 22, 245-57 (2006).

111.

Gehrig, K., Cornell, R.B. & Ridgway, N.D. Expansion of the nucleoplasmic reticulum requires the coordinated activity of lamins and CTP:phosphocholine cytidylyltransferase alpha. Mol Biol Cell 19, 237-47 (2008).

112.

Wang, Y., Sweitzer, T.D., Weinhold, P.A. & Kent, C. Nuclear localization of soluble CTP:phosphocholine cytidylyltransferase. Journal of Biological Chemistry 268, 5899-904 (1993).

113.

Butterworth, M.B. et al. The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel. J Biol Chem 282, 37885-93 (2007).

114.

Wojcik, C. & DeMartino, G.N. Intracellular localization of proteasomes. Int J Biochem Cell Biol 35, 579-89 (2003).

204 115.

Nikko, E. & Andre, B. Evidence for a direct role of the Doa4 deubiquitinating enzyme in protein sorting into the MVB pathway. Traffic 8, 566-81 (2007).

116.

Wang, Y. & Kent, C. Effects of altered phosphorylation sites on the properties of CTP:phosphocholine cytidylyltransferase. J Biol Chem 270, 17843-9 (1995).

117.

Winston, J.T., Koepp, D.M., Zhu, C., Elledge, S.J. & Harper, J.W. A family of mammalian F-box proteins. Curr Biol 9, 1180-2 (1999).

118.

Ho, M.S., Tsai, P.I. & Chien, C.T. F-box proteins: the key to protein degradation. J Biomed Sci 13, 181-91 (2006).

119.

Sharfi, H. & Eldar-Finkelman, H. Sequential phosphorylation of insulin receptor substrate-2 by glycogen synthase kinase-3 and c-Jun NH2-terminal kinase plays a role in hepatic insulin signaling. Am J Physiol Endocrinol Metab 294, E307-15 (2008).

120.

Mahalakshmi, R.N. et al. Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport. Traffic 8, 1164-78 (2007).

121.

Vary, T.C., Deiter, G. & Kimball, S.R. Phosphorylation of eukaryotic initiation factor eIF2Bepsilon in skeletal muscle during sepsis. Am J Physiol Endocrinol Metab 283, E1032-9 (2002).

122.

Maine, G.N., Mao, X., Komarck, C.M. & Burstein, E. COMMD1 promotes the ubiquitination of NF-kappaB subunits through a cullin-containing ubiquitin ligase. Embo J 26, 436-47 (2007).

123.

Larbaud, D. et al. Differential regulation of the lysosomal, Ca2+-dependent and ubiquitin/proteasome-dependent proteolytic pathways in fast-twitch and slowtwitch rat muscle following hyperinsulinaemia. Clin Sci (Lond) 101, 551-8 (2001).

124.

Massey, M., Algar, W.R. & Krull, U.J. Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Forster distances for various dyeDNA conjugates. Anal Chim Acta 568, 181-9 (2006).

125.

Perkins, D.N., Pappin, D.J., Creasy, D.M. & Cottrell, J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551-67 (1999).

126.

Xiao, G.G. et al. Identification of F-box/LLR-repeated protein 17 as potential useful biomarker for breast cancer therapy. Cancer Genomics Proteomics 5, 15160 (2008).

127.

Ravindran, A. et al. Calmodulin-dependent gating of Cav1.2 calcium channels in the absence of Cav{beta} subunits. Proc Natl Acad Sci U S A (2008).

205 128.

Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882-7 (1997).

129.

Sarikas, A., Xu, X., Field, L.J. & Pan, Z.Q. The cullin7 E3 ubiquitin ligase: A novel player in growth control. Cell Cycle 7(2008).

130.

Zhang, Y. et al. F-box Protein DOR Functions as a Novel Inhibitory Factor for ABA-induced Stomatal Closure under Drought Stress in Arabidopsis thaliana. Plant Physiol (2008).

131.

Ohsaki, K. et al. The role of beta-TrCP1 and beta-TrCP2 in circadian rhythm generation by mediating degradation of clock protein PER2. J Biochem (2008).

132.

Ishikawa, Y., Onoyama, I., Nakayama, K.I. & Nakayama, K. Notch-dependent cell cycle arrest and apoptosis in mouse embryonic fibroblasts lacking Fbxw7. Oncogene 27, 6164-74 (2008).

133.

Ilyin, G.P., Rialland, M., Glaise, D. & Guguen-Guillouzo, C. Identification of a novel Skp2-like mammalian protein containing F-box and leucine-rich repeats. FEBS Lett 459, 75-9 (1999).

134.

Frescas, D. & Pagano, M. Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer. Nat Rev Cancer 8, 438-49 (2008).

135.

Kraetke, O. et al. Dimerization of corticotropin-releasing factor receptor type 1 is not coupled to ligand binding. J Recept Signal Transduct Res 25, 251-76 (2005).

136.

Miyawaki, A., Griesbeck, O., Heim, R. & Tsien, R.Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci U S A 96, 2135-40 (1999).

137.

Han, J.Y. et al. C-jun N-terminal kinase regulates the interaction between 14-3-3 and Bad in ethanol-induced cell death. J Neurosci Res 86, 3221-9 (2008).

138.

Aitken, A., Jones, D., Soneji, Y. & Howell, S. 14-3-3 proteins: biological function and domain structure. Biochem Soc Trans 23, 605-11 (1995).

139.

van Heusden, G.P. 14-3-3 proteins: regulators of numerous eukaryotic proteins. IUBMB Life 57, 623-9 (2005).

140.

Xiao, B. et al. Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways. Nature 376, 188-91 (1995).

141.

Ryan, A.J., Andrews, M., Zhou, J. & Mallampalli, R.K. c-Jun N-terminal kinase regulates CTP:phosphocholine cytidylyltransferase. Arch Biochem Biophys 447, 23-33 (2006).

206 142.

Lagace, T.A., Miller, J.R. & Ridgway, N.D. Caspase processing and nuclear export of CTP:phosphocholine cytidylyltransferase alpha during farnesol-induced apoptosis. Mol Cell Biol 22, 4851-62 (2002).