Regulation and Function of PP2A Family Phosphatases 公开
Hwang, Juyeon (2015)
Abstract
PP2A is a multifunctional serine/threonine protein phosphatase that is critical for a variety of cellular processes and thus its dysfunction has been linked to human diseases including cancer and Alzheimer's disease (AD). PP2A is a heterotrimeric enzyme composed of a structural A subunit, a catalytic C (PP2Ac) subunit and one of many regulatory B-type subunits. Reversible carboxyl methylation of PP2Ac on its C-terminal leucine residue by leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) differentially regulates the binding of certain B-type subunits and thus PP2A heterotrimer formation and function. While the PP2A family phosphatases, PP4 and PP6, possess the conserved C-terminal leucine residue, only PP4c has been shown to be methylated before. In Chapter 2.1, I hypothesized that LCMT-1 is the major methyltransferase for both PP4 and PP6, and that methylation of PP4c and PP6c regulates holoenzyme assembly of PP4 and PP6. Results indicated that PP4c and PP6c are highly methylated on their C-termini and that LCMT-1 is the major methyltransferase responsible for methylating these phosphatases. In addition, blocking PP4c methylation by LCMT-1 knockout resulted in loss of methylation-dependent PP4 holoenzyme complexes. Consistent with this, phosphorylation of PP4R1 substrate, HDAC3, was increased by LCMT-1 loss. These results support the hypothesis that carboxyl methylation of PP2A family phosphatase C subunits is a general strategy for regulating holoenzyme assembly and function of these phosphatases. In Chapter 2.2, my objective was to determine the molecular organization of PP2A complexes containing the striatin family members as their B-type subunits. This novel, striatin-associated PP2A complex has been recently implicated in human diseases, especially cerebral cavernous malformations (CCM). Using a structure-function analysis, I found that striatin-associated PP2A regulates the phosphorylation and activation of Mst3 kinase. Finally, in Chapter 2.3, I found that inhibition of PP2A by okadaic acid increased recruitment of Mst3 and Mst4 kinases to striatin. Altogether, these studies provide insights into the regulation of PP2A family phosphatase holoenzyme assembly and function by methylation. Additionally, these studies also provide further insights into the molecular organization and function of striatin-associated PP2A complexes.
Table of Contents
Chapter 1: General Introduction. 1
Protein phosphatase 2A (PP2A). 1
PP2A structure. 1
The PP2Ac C-terminus and PP2A holoenzyme assembly. 3
Carboxyl methylation of PP2Ac. 4
PP2A and human diseases. 9
PP2A-related protein phosphatases. 11
Protein phosphatase 4 (PP4). 11
PP4 regulatory subunits (PP4Rs) and functions. 13
Protein phosphatase 6 (PP6). 18
One common function of PP2A family phosphatases as a g-H2AX phosphatase. 19
PP4 and PP6 in human diseases. 22
Alpha4 (a4): a common binding partner of PP2A family phosphatases. 23
The striatin family of proteins. 27
Striatin. 27
SG2NA. 29
Zinedin. 30
Domain structure of the striatin family proteins. 30
The caveolin-binding domain. 30
The coiled-coil domain. 33
The Ca2+-calmodulin (CaM)-binding domain. 33
The WD-repeat domain. 34
STRIPAK (Striatin-interacting phosphatase and kinase) complexes. 35
Negative regulation of kinases by STRIPAK-associated PP2A. 37
STRIPAK and vesicular trafficking. 39
STRIPAK and cerebral cavernous malformations (CCM) disease. 40
Ccm3 functions and their relation to CCM pathogenesis. 41
Goals of the Dissertation. 43
CHAPTER 2: RESULTS. 48
Carboxyl Methylation of Protein Phosphatase 4 by LCMT-1 is important for PP4 holoenzyme assembly and function. 48
Introduction. 48
Methods and materials. 51
Antibodies and other reagents. 51
Cell culture and transfection. 52
Cell lysis, immunoprecipitation, SDS-PAGE, and immunoblotting. 52
Methylation assay. 53
Blue native polyacrylamide gel electrophoresis (BN-PAGE). 53
Results. 54
LCMT-1 is the major methyltransferase for PP4c and PP6c. 54
Loss of LCMT-1 affects formation of PP2A holoenzyme complexes. 59
Loss of PP2Ac methylation by LCMT-1 knockout redistributes PP2A complexes: Loss of PP2Ac methylation by LCMT-1 knockout increases co-migration of a4 with PP2Ac. 63
LCMT-1-mediated PP4 methylation regulates the stable formation of certain PP4 complexes. 65
PP4R1-associated PP4 holoenzyme complex is methylation-dependent. 68
The methylation-dependent PP4R1-associated PP4 holoenzyme complex is largely disrupted by LCMT-1 knockout. 73
Loss of LCMT-1 results in hyperphosphorylation and activation of HDAC3. 75
Discussion. 75
CHAPTER 3: RESULTS. 83
Protein Phosphatase 2A (PP2A) Binds Within the Oligomerization Domain of Striatin and Regulates the Phosphorylation and Activation of the Mammalian Ste20-Like Kinase Mst3. 83
Abstract. 84
Background. 85
Results. 86
Generation of striatin mutants for structure-function analysis of striatin complexes. 86
The WD-repeats of striatin are not required for PP2A binding but contribute to Mob3 association. 86
Both N- and C-terminal sequences of striatin associate with Mob3 but only N-terminal striatin sequences associate stably with PP2A. 86
The coiled-coil and caveolin-binding domains of striatin, but not the calmodulin-binding domain of striatin, are necessary for oligomerization and for PP2A binding. 87
Residues in the coiled-coil domain of striatin are critical for PP2A C subunit association independent from their role in oligomerization. 89
Determinants within striatin residues 191-344 are critical for binding to Mst3 and Mst4 kinases and to CCM3. 89
The calmodulin-binding domain of striatin negatively regulates association with the Mst3 and Mst4 kinases. 91
Striatin-associated PP2A negatively regulates the phosphorylation of Mst3 kinase. 92
PP2A negatively regulates the activation of striatin-associated Mst3 kinase. 94
Discussion. 94
Conclusions. 97
Methods. 98
Antibodies. 98
Plasmids, mutagenesis, and creation of stable cell lines. 98
Cell culture, transfection, and cell lysis. 98
Immunoprecipitation, gel electrophoresis, and immunoblotting. 98
Phosphorylation of Mst3 in vivo. 99
Dephosphorylation of Mst3 in vitro. 99
Immunoprecipitation of denatured lysates. 99
References. 100
Chapter 4: ADDITIONAL RESULTS. 102
PP2A negatively regulates the recruitment of Mst3 and Mst4 to striatin complexes. 102
Increased binding of Mst3 and Mst4 to striatin upon inhibition of PP2A by okadaic acid. 102
Discussion. 102
CHAPTER 5: DISCUSSION and FUTURE DIRECTIONS. 106
PME-1, PME-1 inhibitors, and cancer. 106
LCMT-1 negatively regulates HDAC3 phosphorylation and activation. 107
Functional significance of the PP4c methylation. 108
Regulation of subcellular localization of the striatin complexes. 1100
Implication of Ccm3-GCKIII and STRIPAK functions in CCM pathogenesis. 110
Overview. 112
CHAPTER 6: REFERENCES. 116
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