Purification and Identification of a Novel Subunit of Protein Serine/Threonine Phosphatase 4

The protein phosphatase 2A (PP2A) subfamily of phosphatases, PP2A, PP4, and PP6, are multifunctional serine/threonine protein phosphatases involved in many cellular processes. Carboxyl methylation of the PP2A catalytic subunit (PP2Ac) C-terminal leucine is regulated by the opposing activities of leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) and regulates PP2A holoenzyme formation. The site of methylation on PP2Ac is conserved in the catalytic subunits of PP4 and PP6, and PP4 is also methylated on that site, but the identities of the methyltransferase enzyme for PP4 are not known. Whether PP6 is methylated is also not known. Here we use antibodies specific for the unmethylated phosphatases to show that PP6 is carboxyl-methylated and that LCMT-1 is the major methyltransferase for PP2A, PP4, and PP6 in mouse embryonic fibroblasts (MEFs). Analysis of PP2A and PP4 complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) indicates that PP4 holoenzyme complexes, like those of PP2A, are differentially regulated by LCMT-1, with the PP4 regulatory subunit 1 (PP4R1)-containing PP4 complex being the most dramatically affected by the LCMT-1 loss. MEFs derived from LCMT-1 knock-out mouse embryos have reduced levels of PP2A B regulatory subunit and PP4R1 relative to control MEFs, indicating that LCMT-1 is important for maintaining normal levels of these subunits. Finally, LCMT-1 homozygous knock-out MEFs exhibited hyperphosphorylation of HDAC3, a reported target of the methylation-dependent PP4R1-PP4c complex. Collectively, our data suggest that LCMT-1 coordinately regulates the carboxyl methylation of PP2A-related phosphatases and, consequently, their holoenzyme assembly and function.

Section: Lcmt-1 Differentially Regulates Different Pp4 Holoenzymessupporting

confidence: 57%

mentioning

confidence: 99%

Leucine Carboxyl Methyltransferase 1 (LCMT-1) Methylates Protein Phosphatase 4 (PP4) and Protein Phosphatase 6 (PP6) and Differentially Regulates the Stable Formation of Different PP4 Holoenzymes

Hwang

Lee²,

Pallas

2016

“…The present studies introduce yet another potential regulatory paradigm for serine/threonine phosphatases by which two recently identified PP2A inhibitors, I 1 PP2A and I 2 PP2A , implicated in human cancers, associated with another major cellular phosphatase, PP1, and enhanced its activity in vitro against selected phosphoprotein substrates in the presence of nearly physiological concentrations of Mn 2ϩ . In this regard, it is noteworthy that PP2A and the structurally related enzymes PP4 and PP6 not only share the property of associating with ␣4 (35) but also associate with a recently identified novel PP4 subunit with structural similarity to the A-subunit (36). Whether PP4 and other related enzymes include domains required for I 1 PP2A and I 2 PP2A binding and whether these two proteins regulate PP4 and other PP2A-related phosphatases remains to be investigated.…”

Section: Discussionmentioning

confidence: 99%

Protein Phosphatase 2A Inhibitors, I1PP2A and I2PP2A, Associate with and Modify the Substrate Specificity of Protein Phosphatase 1

Katayose

Al-Murrani

et al. 2000

“…Immunopurification, Western Blot, and Gel Analysis-The general serine/threonine phosphatase antibody (FL-18) was from Santa Cruz Biotechnology. Antibodies to OGT, PP1␣, PP1␤, PP1␥, PP4, and PP6 have been described previously (12,(22)(23)(24)(25)(26)(27). Negative controls used normal rabbit IgG.…”

Section: Methodsmentioning

confidence: 99%

O-GlcNAc Transferase Is in a Functional Complex with Protein Phosphatase 1 Catalytic Subunits

Wells

Kreppel

Comer

et al. 2004

Self Cite

128

113

A hallmark of signal transduction is the dynamic and inducible post-translational modification of proteins. In addition to the well characterized phosphorylation of proteins, other modifications have been shown to be regulatory, including O-linked ␤-N-acetylglucosamine (O-GlcNAc). O-GlcNAc modifies serine and threonine residues on a myriad of nuclear and cytosolic proteins, and for several proteins there appears to be a reciprocal relationship between phosphorylation and O-GlcNAc modification. Here we report further evidence of this yin-yang relationship by demonstrating that O-GlcNAc transferase, the enzyme that adds O-GlcNAc to proteins, exists in stable and active complexes with the serine/ threonine phosphatases PP1␤ and PP1␥, enzymes that remove phosphate from proteins. The existence of this complex highlights the importance of understanding the dynamic relationship between O-GlcNAc and phosphate in modulating protein function in many cellular processes and disease states such as Alzheimer's disease and type II diabetes.Although there are only 21, counting selenocysteine, genomically encoded mammalian amino acids, post-translational modification results in proteins that contain more than 50 different amino acids (1). Thus, covalent modification of polypeptides plays a major role in the generation of functional polypeptides and adds an extra level of complexity in attempts to understand the proteome (2). While several protein modifications are static, a subset of post-translational modifications is both dynamic and inducible. It is the study of these "regulatory" posttranslational modifications and the enzymes that add and subtract them to proteins that comprises a significant portion of the signal transduction field (3).The most well studied and understood regulatory post-translational modification is phosphorylation (3). Accordingly, a significant body of literature has focused on the kinase superfamily, which makes up more than 2% of the proteins encoded by the human genome (4). Interestingly, in humans there appear to be only about 15 catalytic serine/threonine protein phosphatases (PPP subfamily) compared with literally hundreds of serine/threonine kinases (4). Thus, understanding how protein dephosphorylation is regulated has been an area of intense study for the last two decades (5, 6). One emerging theme is that the catalytic phosphatases have binding partners that regulate their localization and activity (7).Similar to phosphorylation, O-linked ␤-N-acetylglucosamine (O-GlcNAc) modification of nuclear and cytosolic proteins is an abundant, dynamic, and inducible post-translational modification (8 -10). However, unlike kinases, there appears to be only one O-GlcNAc transferase catalytic subunit (OGT, 1 the enzyme that adds O-GlcNAc to proteins) in mammals (11). Similar to phosphatases, it has been proposed that OGT is regulated by a multitude of binding partners as well as post-translational modification and alternative splicing (12, 13). It has been demonstrated recently that OGT interacts with sever...