title: Leucine methylation of protein phosphatase PP4C at C-terminal is critical for its cellular functions.
abstract: BACKGROUND
  Protein phosphatase 4 (PP4) has been known to have critical functions in DNA double strand break (DSB) repair and cell cycle by the regulation of phosphorylation of its target proteins, such as H2AX, RPA2, KAP-1, 53BP1. However, it is largely unknown how PP4 itself is regulated.
  
  
  METHODS
  We examined the PP4C methylation on L307 at C-terminal by using methylated-leucine specific antibody. Then with PP4C L307A mutant, we explored that how nonmethylated form of PP4C affects its known cellular functions by immunoprecipitation, immunofluorescence, and DNA DSB repair assays.
  
  
  RESULTS
  Here we show that PP4C is methylated on its C-terminal leucine residue in vivo and this methylation is important for cellular functions mediated by PP4. In the cells PP4C L307A mutant has significantly low activity of dephosphorylation against its known target proteins, and the loss of interaction between L307A PP4 mutant and regulatory subunits, R1, R2, or R3α/β causes the dissociation from its target proteins. Moreover, PP4C L307A mutant loses its role in both DSB repair pathways, HR (homologous recombination) and NHEJ (non-homologous end joining), which phenocopies PP4C depletion.
  
  
  CONCLUSIONS
  Our results demonstrate the key site of PP4C methylation and establish the physiological importance of this regulation.
authors.affiliation: Department of Biological Sciences, College of Science, Chonnam National University, Gwangju 500-757, Republic of Korea.
authors.forename: JungJin
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, College of Science, Chonnam National University, Gwangju 500-757, Republic of Korea. Electronic address: donghyunlee73@jnu.ac.kr.
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D008745:Methylation; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering
chemical_list: D034741:RNA, Small Interfering; D010749:Phosphoprotein Phosphatases; C080406:protein phosphatase 4; D007930:Leucine
keywords: DNA damage response; Double-strand DNA breaks repair; Homologous recombination; Leucine methylation; Non-homologous end joining; Protein phosphatase 4