Leucine methylation of protein phosphatase PP4C at C-terminal is critical for its cellular functions

Lee, Jungjin; Lee, Dong-Hyun

doi:10.1016/j.bbrc.2014.08.045

Cited by 12 publications

(12 citation statements)

References 25 publications

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“…In contrast to our findings reported here that methylation of Leu-307 in PP4c has differential effects on PP4 complexes, a recent study using a mutational approach to explore the function of PP4c methylation concluded that loss of PP4c methylation has a severe global effect on PP4 complexes and function (52). In that study the authors changed the C-terminal leucine (Leu-307) of PP4c to alanine (L307A mutant), which alters the side chain of the C-terminal amino acid and may affect methylation.…”

Section: Lcmt-1 Differentially Regulates Different Pp4 Holoenzymescontrasting

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

Journal of Biological Chemistry

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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.

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Section: Lcmt-1 Differentially Regulates Different Pp4 Holoenzymescontrasting

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

Journal of Biological Chemistry

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“…To determine whether PP4C can dephosphorylate pT454-DBC1 directly, we immunopurified phospho-DBC1 from IR-treated cells and performed dephosphorylation assays as described earlier ( Lee et al, 2014 ; Smith et al, 2010 ). PP4C dephosphorylates pT454-DBC1 in a dose-dependent manner ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The in vitro dephosphorylation assay was performed as described ( Lee and Lee, 2014 ; Lee et al, 2012 ). PP4C WT and D82A mutant proteins were purified using the Bac-to-Bac Baculovirus Expression System (Invitrogen) according to the manufacturer’s manual.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we and others have identified the role of the protein phosphatase 4 (PP4) in DDR ( Chowdhury et al, 2008 ; Lee and Lee, 2014 ; Lee et al, 2010 ; 2012 ; 2014 ; Nakada et al, 2008 ; Wang et al, 2008 ). PP4 dephosphorylated the essential proteins, including replication protein A 2 (RPA2), KAP-1, and 53BP1 after DNA damage and these dephosphorylation events were critical for the efficient repair of DNA double-strand breaks (DSBs) ( Lee and Lee, 2014 ; Lee et al, 2010 ; 2012 ; 2014 ). To study the functions of PP4 in depth, we performed tandem affinity purification followed by mass spectrometry.…”

Section: Introductionmentioning

confidence: 99%

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Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions

Lee¹,

Adelmant²,

Marto³

et al. 2015

Molecules and Cells

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Deleted in breast cancer-1 (DBC1) contributes to the regulation of cell survival and apoptosis. Recent studies demonstrated that DBC is phosphorylated at Thr454 by ATM/ATR kinases in response to DNA damage, which is a critical event for p53 activation and apoptosis. However, how DBC1 phosphorylation is regulated has not been studied. Here we show that protein phosphatase 4 (PP4) dephosphorylates DBC1, regulating its role in DNA damage response. PP4R2, a regulatory subunit of PP4, mediates the interaction between DBC1 and PP4C, a catalytic subunit. PP4C efficiently dephosphorylates pThr454 on DBC1 in vitro, and the depletion of PP4C/PP4R2 in cells alters the kinetics of DBC1 phosphorylation and p53 activation, and increases apoptosis in response to DNA damage, which are compatible with the expression of the phosphomimetic DBC-1 mutant (T454E). These suggest that the PP4-mediated dephosphorylation of DBC1 is necessary for efficient damage responses in cells.

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“…These phosphatases are structurally and evolutionarily related to Protein Phosphatase 1 (PP1). They share particular distinctive characteristics such as sensitivity to inhibition by okadaic acid in the nanomolar range and the presence of a conserved C-terminal motif (PDYFL) that is subject to post-translational regulation in PP2Ac 1 2 and possibly also in PP4c and PP6c 3 . PP2A is a major regulator of several cellular processes and an important tumor suppressor.…”

mentioning

confidence: 99%

Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus

Scorsato

Lima

Righetto

et al. 2016

Sci Rep

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TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15 Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/β region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase’s active site, providing a structural framework for the function of TIPRL in PP2A inhibition.

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Leucine methylation of protein phosphatase PP4C at C-terminal is critical for its cellular functions

Cited by 12 publications

References 25 publications

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

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

Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions

Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus

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