title: An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria.
abstract: A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchoring proteins also bind the type I (RI) regulatory subunit of PKA with 10-100-fold lower affinity. A range of cellular, biochemical, biophysical, and genetic approaches comprehensively establish that sphingosine kinase interacting protein (SKIP) is a truly type I-specific AKAP. Mapping studies located anchoring sites between residues 925-949 and 1,140-1,175 of SKIP that bind RI with dissociation constants of 73 and 774 nM, respectively. Molecular modeling and site-directed mutagenesis approaches identify Phe 929 and Tyr 1,151 as RI-selective binding determinants in each anchoring site. SKIP complexes exist in different states of RI-occupancy as single-molecule pull-down photobleaching experiments show that 41 ± 10% of SKIP sequesters two YFP-RI dimers, whereas 59 ± 10% of the anchoring protein binds a single YFP-RI dimer. Imaging, proteomic analysis, and subcellular fractionation experiments reveal that SKIP is enriched at the inner mitochondrial membrane where it associates with a prominent PKA substrate, the coiled-coil helix protein ChChd3.
authors.affiliation: Department of Pharmacology, Howard Hughes Medical Institute, University of Washington School of Medicine, 1959 Pacific Avenue NE, Seattle, WA 98195, USA.
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Means
authors.forename: Birgitte
authors.initials: B
authors.lastname: Lygren
authors.forename: Lorene K
authors.initials: LK
authors.lastname: Langeberg
authors.forename: Ankur
authors.initials: A
authors.lastname: Jain
authors.forename: Rose E
authors.initials: RE
authors.lastname: Dixon
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Vega
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Gold
authors.forename: Susanna
authors.initials: S
authors.lastname: Petrosyan
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
authors.forename: Anne N
authors.initials: AN
authors.lastname: Murphy
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
authors.forename: Luis F
authors.initials: LF
authors.lastname: Santana
authors.forename: Kjetil
authors.initials: K
authors.lastname: Tasken
authors.forename: John D
authors.initials: JD
authors.lastname: Scott
mesh_terms: D054758:A Kinase Anchor Proteins / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D020349:Surface Plasmon Resonance; D014162:Transfection
chemical_list: D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; C521110:CHCHD3 protein, human; D024101:Mitochondrial Proteins; C466999:SPHKAP protein, human; D017868:Cyclic AMP-Dependent Protein Kinases
references.citation: Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1627-32
references.pmid: 12571360
references.citation: Protein Eng Des Sel. 2011 Mar;24(3):333-9
references.pmid: 21115539
references.citation: Am J Physiol Heart Circ Physiol. 2007 Jun;292(6):H2944-51
references.pmid: 17293497
references.citation: J Immunol. 2007 Oct 15;179(8):5159-68
references.pmid: 17911601
references.citation: Nature. 2007 Dec 13;450(7172):973-82
references.pmid: 18075576
references.citation: Trends Cell Biol. 1999 Jun;9(6):216-21
references.pmid: 10354567
references.citation: Biochim Biophys Acta. 2004 Mar 11;1697(1-2):259-69
references.pmid: 15023366
references.citation: J Biol Chem. 1997 Mar 21;272(12):8057-64
references.pmid: 9065479
references.citation: J Biol Chem. 2008 May 2;283(18):11954-63
references.pmid: 18296752
references.citation: J Cell Biol. 2007 Feb 12;176(4):521-33
references.pmid: 17296797
references.citation: Science. 2002 Dec 6;298(5600):1912-34
references.pmid: 12471243
references.citation: Nat Rev Mol Cell Biol. 2004 Dec;5(12):959-70
references.pmid: 15573134
references.citation: Cell. 1995 Jan 27;80(2):225-36
references.pmid: 7834742
references.citation: EMBO J. 2001 Apr 2;20(7):1651-62
references.pmid: 11285229
references.citation: J Biol Chem. 1986 Sep 15;261(26):12352-61
references.pmid: 2427518
references.citation: Pharmacol Ther. 1991;50(1):123-45
references.pmid: 1653962
references.citation: Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4445-50
references.pmid: 12672969
references.citation: EMBO J. 2003 Oct 15;22(20):5491-500
references.pmid: 14532121
references.citation: J Mol Biol. 2003 Jul 25;330(5):1117-29
references.pmid: 12860132
references.citation: Structure. 2010 Feb 10;18(2):155-66
references.pmid: 20159461
references.citation: J Biol Chem. 1991 Dec 5;266(34):23074-82
references.pmid: 1744104
references.citation: J Biol Chem. 1977 Jun 10;252(11):3854-61
references.pmid: 16921
references.citation: Mol Interv. 2010 Apr;10(2):86-97
references.pmid: 20368369
references.citation: J Biol Chem. 2002 Sep 20;277(38):35257-62
references.pmid: 12124383
references.citation: J Biol Chem. 2011 Jan 28;286(4):2918-32
references.pmid: 21081504
references.citation: J Biol Chem. 2008 Nov 28;283(48):33708-18
references.pmid: 18824551
references.citation: Proc Natl Acad Sci U S A. 1987 Aug;84(15):5192-6
references.pmid: 3037538
references.citation: Proc Natl Acad Sci U S A. 1983 Jun;80(12):3608-12
references.pmid: 6190178
references.citation: Chembiochem. 2010 May 3;11(7):963-71
references.pmid: 20394097
references.citation: J Biol Chem. 1977 Jul 25;252(14):4888-94
references.pmid: 194899
references.citation: Nat Rev Genet. 2010 May;11(5):345-55
references.pmid: 20376054
references.citation: EMBO J. 1999 Apr 1;18(7):1858-68
references.pmid: 10202149
references.citation: J Biol Chem. 1990 Dec 15;265(35):21561-6
references.pmid: 2147685
references.citation: Am J Physiol Cell Physiol. 2010 Jun;298(6):C1343-52
references.pmid: 20357183
references.citation: Nature. 1984 Nov 15-21;312(5991):237-42
references.pmid: 6504138
references.citation: J Mol Endocrinol. 2010 May;44(5):271-84
references.pmid: 20150326
references.citation: J Biol Chem. 2001 Nov 23;276(47):44247-57
references.pmid: 11546812
references.citation: Science. 1998 Jan 23;279(5350):509-14
references.pmid: 9438836
references.citation: Dev Biol. 2000 Jul 1;223(1):194-204
references.pmid: 10864471
references.citation: Methods Enzymol. 1974;38:287-90
references.pmid: 4375761
references.citation: J Biol Chem. 2005 Jul 15;280(28):26011-7
references.pmid: 15883165
references.citation: Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11184-9
references.pmid: 9326583
references.citation: Mol Cell. 2011 Apr 8;42(1):84-95
references.pmid: 21474070
references.citation: Mol Cell. 2006 Nov 3;24(3):383-95
references.pmid: 17081989
references.citation: Mol Cell. 2006 Nov 3;24(3):397-408
references.pmid: 17081990
references.citation: J Biol Chem. 2000 Nov 10;275(45):35146-52
references.pmid: 10899163
references.citation: J Biol Chem. 1991 Aug 5;266(22):14188-92
references.pmid: 1860836
references.citation: J Biol Chem. 1975 Jan 10;250(1):218-25
references.pmid: 166986
references.citation: Circ Res. 2003 Mar 7;92(4):342-4
references.pmid: 12623870
references.citation: Curr Opin Neurobiol. 1997 Jun;7(3):397-403
references.pmid: 9232801
references.citation: Proc Natl Acad Sci U S A. 1986 Mar;83(6):1613-6
references.pmid: 3456605
references.citation: Methods Enzymol. 1974;38:299-308
references.pmid: 4375763
references.citation: Proc Natl Acad Sci U S A. 2011 Apr 19;108(16):6426-31
references.pmid: 21464287
references.citation: J Biol Chem. 2002 Jul 26;277(30):27294-304
references.pmid: 12004056
references.citation: J Proteome Res. 2006 Jun;5(6):1435-47
references.pmid: 16739995
references.citation: J Biol Chem. 2007 May 18;282(20):14952-9
references.pmid: 17242405
references.citation: Science. 2009 Nov 27;326(5957):1220-4
references.pmid: 19965465
references.citation: Nat Rev Mol Cell Biol. 2002 Sep;3(9):710-8
references.pmid: 12209131
references.citation: J Biol Chem. 2002 Sep 6;277(36):32947-53
references.pmid: 12080051
references.citation: Nature. 2011 May 26;473(7348):484-8
references.pmid: 21614075
references.citation: Biochem J. 2006 Dec 15;400(3):493-9
references.pmid: 16948636