title: p21-Activated kinase 1 (Pak1) phosphorylates BAD directly at serine 111 in vitro and indirectly through Raf-1 at serine 112.
abstract: BACKGROUND
  Cell survival depends on the balance between protective and apoptotic signals. When the balance of signals tips towards apoptosis, cells undergo programmed cell death. This balance has profound implications in diseases including cancer. Oncogenes and tumor suppressors are mutated to promote cell survival during tumor development, and many chemotherapeutic drugs kill tumor cells by stimulating apoptosis. BAD is a pro-apoptotic member of the Bcl-2 family of proteins, which can be phosphorylated on numerous sites to modulate binding to Bcl-2 and 14-3-3 proteins and inhibit its pro-apoptotic activities. One of the critical phosphorylation sites is the serine 112 (S112), which can be phosphorylated by several kinases including Pak1.
  
  
  RESULTS
  We mapped the Pak phosphorylation sites by making serine to alanine mutations in BAD and testing them as substrates in in vitro kinase assays. We found that the primary phosphorylation site is not S112 but serine 111 (S111), a site that is sometimes found phosphorylated in vivo. In transfection assays of HEK293T cells, we showed that Pak1 required Raf-1 to stimulate phosphorylation on S112. Mutating either S111 or S112 to alanine enhanced binding to Bcl-2, but the double mutant S111/112A bound better to Bcl-2. Moreover, BAD phosphorylation at S111 was observed in several other cell lines, and treating one of them with the Pak1 inhibitor 2,2'-Dihydroxy-1,1'-dinaphthyldisulfide (IPA-3) reduced phosphorylation primarily at S112 and to a smaller extent at S111, while Raf inhibitors only reduced phosphorylation at S112.
  
  
  CONCLUSIONS
  Together, these findings demonstrate that Pak1 phosphorylates BAD directly at S111, but phosphorylated S112 through Raf-1. These two sites of BAD serve as redundant regulatory sites for Bcl-2 binding.
authors.affiliation: Department of Pharmacology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: Diana Z
authors.initials: DZ
authors.lastname: Ye
authors.forename: Shenghao
authors.initials: S
authors.lastname: Jin
authors.forename: Ya
authors.initials: Y
authors.lastname: Zhuo
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Field
mesh_terms: D002460:Cell Line; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D010766:Phosphorylation; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D051025:bcl-Associated Death Protein / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000378:metabolism*
chemical_list: D051025:bcl-Associated Death Protein; D012694:Serine; D019908:Proto-Oncogene Proteins c-raf; D054462:p21-Activated Kinases
references.citation: Nat Cell Biol. 1999 Dec;1(8):E209-16
references.pmid: 10587660
references.citation: Annu Rev Biochem. 2003;72:743-81
references.pmid: 12676796
references.citation: J Biol Chem. 2005 Jul 1;280(26):24698-705
references.pmid: 15849194
references.citation: Mol Cell Biol. 2000 Jan;20(2):453-61
references.pmid: 10611223
references.citation: Cancer Metastasis Rev. 2009 Jun;28(1-2):51-63
references.pmid: 19165420
references.citation: Cell. 2010 Jan 22;140(2):209-21
references.pmid: 20141835
references.citation: Biochem J. 2000 Jul 15;349(Pt 2):547-57
references.pmid: 10880354
references.citation: Mol Cell Biol. 1999 Mar;19(3):1881-91
references.pmid: 10022875
references.citation: Cell. 1996 Nov 15;87(4):619-28
references.pmid: 8929531
references.citation: Mol Cell Biol. 2004 Jul;24(14):6205-14
references.pmid: 15226424
references.citation: J Biol Chem. 2002 Oct 25;277(43):40944-9
references.pmid: 12189144
references.citation: Mol Cell. 1999 Apr;3(4):413-22
references.pmid: 10230394
references.citation: Curr Biol. 1999 Apr 8;9(7):369-72
references.pmid: 10209122
references.citation: Proc Natl Acad Sci U S A. 2010 May 18;107(20):9446-51
references.pmid: 20439741
references.citation: Chem Biol. 2008 Apr;15(4):322-31
references.pmid: 18420139
references.citation: Neoplasia. 2005 Jul;7(7):638-45
references.pmid: 16026643
references.citation: J Biol Chem. 2001 May 18;276(20):16624-34
references.pmid: 11278362
references.citation: EMBO J. 2001 Apr 17;20(8):1940-51
references.pmid: 11296227
references.citation: Mol Cell Biol. 2001 Apr;21(7):2324-36
references.pmid: 11259582
references.citation: Mol Cell. 2010 Feb 12;37(3):299-310
references.pmid: 20159550
references.citation: Cell. 1997 Oct 17;91(2):231-41
references.pmid: 9346240
references.citation: Proteomics. 2004 Jun;4(6):1551-61
references.pmid: 15174125
references.citation: Mol Cell. 2004 Feb 13;13(3):329-40
references.pmid: 14967141
references.citation: J Biol Chem. 2009 Oct 9;284(41):28004-28020
references.pmid: 19667065
references.citation: Cell. 1996 Nov 15;87(4):629-38
references.pmid: 8929532
references.citation: J Cell Sci. 2002 Apr 15;115(Pt 8):1575-81
references.pmid: 11950876
references.citation: Nature. 2010 Mar 18;464(7287):427-30
references.pmid: 20179705
references.citation: Science. 1999 Nov 12;286(5443):1358-62
references.pmid: 10558990
references.citation: Science. 2003 Jul 4;301(5629):94-6
references.pmid: 12843393
references.citation: J Biol Chem. 2000 Aug 18;275(33):25865-9
references.pmid: 10837486
references.citation: J Cell Sci. 2004 Sep 1;117(Pt 19):4343-54
references.pmid: 15331659
references.citation: J Biol Chem. 2007 May 25;282(21):15667-78
references.pmid: 17392278
references.citation: Mol Cell. 2000 Jul;6(1):41-51
references.pmid: 10949026
references.citation: Mol Cell. 2002 May;9(5):1005-16
references.pmid: 12049737
references.citation: J Biol Chem. 2000 Mar 31;275(13):9106-9
references.pmid: 10734042
references.citation: Nature. 2010 Mar 18;464(7287):431-5
references.pmid: 20130576
references.citation: Nat Rev Cancer. 2006 Jun;6(6):459-71
references.pmid: 16723992
references.citation: Nature. 1994 Jan 6;367(6458):40-6
references.pmid: 8107774
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3635-41
references.pmid: 12824383
references.citation: Cell Death Differ. 2002 Aug;9(8):783-5
references.pmid: 12107820
references.citation: J Cell Biochem. 2008 Sep 1;105(1):167-75
references.pmid: 18465753
references.citation: Biochem Pharmacol. 2003 Oct 15;66(8):1341-5
references.pmid: 14555207
references.citation: Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5139-44
references.pmid: 9560242
references.citation: Nat Genet. 1997 Jul;16(3):293-7
references.pmid: 9207797
references.citation: Mol Pharmacol. 2001 Dec;60(6):1325-31
references.pmid: 11723239
references.citation: Mol Cell Biol. 2003 Aug;23(16):5526-39
references.pmid: 12897128
references.citation: J Biol Chem. 2001 Apr 27;276(17):14414-9
references.pmid: 11278822
references.citation: Nature. 2010 Mar 18;464(7287):358-9
references.pmid: 20237552
references.citation: EMBO J. 2001 Apr 17;20(8):1952-62
references.pmid: 11296228