title: Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners.
abstract: Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P₃ that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P₃ headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P₃ headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P₃-binding proteins.
authors.affiliation: Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, United States.
authors.forename: Meng M
authors.initials: MM
authors.lastname: Rowland
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Bostic
authors.forename: Denghuang
authors.initials: D
authors.lastname: Gong
authors.forename: Anna E
authors.initials: AE
authors.lastname: Speers
authors.forename: Nathan
authors.initials: N
authors.lastname: Lucas
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
authors.forename: Michael D
authors.initials: MD
authors.lastname: Best
mesh_terms: D000480:Alkynes / Q000737:chemistry; D045744:Cell Line, Tumor; D057930:Click Chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008545:Melanoma / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism*; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry*; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D053719:Tandem Mass Spectrometry
chemical_list: D000480:Alkynes; D005456:Fluorescent Dyes; D008024:Ligands; D008565:Membrane Proteins; D015335:Molecular Probes; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D018129:Phosphatidylinositol Phosphates; D019920:Photoaffinity Labels; C060974:phosphatidylinositol 3,4,5-triphosphate; D051057:Proto-Oncogene Proteins c-akt
references.citation: Nat Rev Mol Cell Biol. 2008 Feb;9(2):99-111
references.pmid: 18216767
references.citation: Eur J Biochem. 1997 Apr 15;245(2):512-9
references.pmid: 9151987
references.citation: Eur J Biochem. 1988 May 16;174(1):95-101
references.pmid: 3259506
references.citation: J Biol Chem. 1999 Dec 31;274(53):37893-900
references.pmid: 10608855
references.citation: J Biol Chem. 1998 May 29;273(22):13375-8
references.pmid: 9593664
references.citation: Science. 2002 Dec 6;298(5600):2002-6
references.pmid: 12471262
references.citation: Science. 1999 May 28;284(5419):1527-30
references.pmid: 10348740
references.citation: Annu Rev Biochem. 2008;77:383-414
references.pmid: 18366325
references.citation: Angew Chem Int Ed Engl. 2009;48(38):6974-98
references.pmid: 19714693
references.citation: Chem Biol. 2009 Jan 30;16(1):3-14
references.pmid: 19171301
references.citation: Mol Cell. 2002 Jan;9(1):95-108
references.pmid: 11804589
references.citation: Chembiochem. 2005 Feb;6(2):291-5
references.pmid: 15578642
references.citation: Anal Chem. 1995 Apr 15;67(8):1426-36
references.pmid: 7741214
references.citation: Nat Prod Rep. 2010 Oct;27(10):1403-30
references.pmid: 20714465
references.citation: J Am Chem Soc. 2010 Mar 17;132(10):3264-5
references.pmid: 20178358
references.citation: Bioorg Med Chem Lett. 2007 Jan 15;17(2):376-8
references.pmid: 17095228
references.citation: Nat Rev Mol Cell Biol. 2001 May;2(5):327-38
references.pmid: 11331907
references.citation: J Proteome Res. 2008 Jan;7(1):266-75
references.pmid: 18173223
references.citation: Curr Opin Genet Dev. 2008 Feb;18(1):97-106
references.pmid: 18294838
references.citation: Mol Cell Proteomics. 2007 Nov;6(11):1829-41
references.pmid: 17617665
references.citation: Nat Prod Rep. 2007 Aug;24(4):687-707
references.pmid: 17653355
references.citation: J Biol Chem. 1998 Nov 13;273(46):30497-508
references.pmid: 9804818
references.citation: Oncogene. 2008 Sep 18;27(41):5497-510
references.pmid: 18794884
references.citation: Cancer Res. 2005 Sep 15;65(18):8339-49
references.pmid: 16166311
references.citation: Bioconjug Chem. 2008 Sep;19(9):1855-63
references.pmid: 18683963
references.citation: Biochemistry. 2009 Jul 21;48(28):6571-84
references.pmid: 19485420
references.citation: Bioorg Med Chem Lett. 1999 Jun 21;9(12):1697-702
references.pmid: 10397504
references.citation: Nat Biotechnol. 2001 Mar;19(3):242-7
references.pmid: 11231557
references.citation: J Biol Chem. 2007 Nov 2;282(44):32093-105
references.pmid: 17823121
references.citation: Science. 2004 Apr 23;304(5670):554
references.pmid: 15016963
references.citation: Nat Rev Mol Cell Biol. 2008 Feb;9(2):162-76
references.pmid: 18216772
references.citation: Org Biomol Chem. 2006 Dec 7;4(23):4387-94
references.pmid: 17102885
references.citation: Science. 1997 Jul 25;277(5325):567-70
references.pmid: 9228007
references.citation: Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10472-6
references.pmid: 7479822
references.citation: Chem Biol. 2004 Nov;11(11):1523-31
references.pmid: 15556003
references.citation: Mol Biosyst. 2009 Sep;5(9):962-72
references.pmid: 19668861
references.citation: Cell. 2008 May 2;133(3):403-14
references.pmid: 18455982
references.citation: J Biol Chem. 1997 Mar 28;272(13):8474-81
references.pmid: 9079675
references.citation: J Proteome Res. 2008 Dec;7(12):5295-313
references.pmid: 19367725
references.citation: Mol Biosyst. 2010 Oct;6(10):1751-9
references.pmid: 20544074
references.citation: Carbohydr Res. 1992 Oct 9;234:65-73
references.pmid: 1468081
references.citation: Annu Rev Biophys Biomol Struct. 2005;34:119-51
references.pmid: 15869386
references.citation: Oncogene. 2004 Mar 4;23(9):1754-65
references.pmid: 14755253
references.citation: FEBS Lett. 2003 Jul 3;546(1):25-31
references.pmid: 12829232
references.citation: J Am Chem Soc. 2003 Mar 19;125(11):3192-3
references.pmid: 12630856
references.citation: Anal Biochem. 2000 May 1;280(2):301-7
references.pmid: 10790314
references.citation: Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10335-40
references.pmid: 12149457
references.citation: Cancer Lett. 2006 Sep 28;241(2):184-96
references.pmid: 16412571
references.citation: Nature. 2006 Oct 12;443(7112):651-7
references.pmid: 17035995
references.citation: J Proteome Res. 2002 Jan-Feb;1(1):21-6
references.pmid: 12643522
references.citation: Chem Biol. 2006 Oct;13(10):1041-50
references.pmid: 17052608
references.citation: Cell. 2007 Jun 29;129(7):1261-74
references.pmid: 17604717
references.citation: Annu Rev Cell Dev Biol. 2001;17:615-75
references.pmid: 11687500
references.citation: FASEB J. 2000 Jun;14(9):1179-87
references.pmid: 10834940
references.citation: Biochem J. 2009 Jan 15;417(2):411-21
references.pmid: 19099539
references.citation: Biochem J. 1990 Dec 15;272(3):817-25
references.pmid: 2176480
references.citation: Nat Chem Biol. 2008 Mar;4(3):203-13
references.pmid: 18246061
references.citation: Bioconjug Chem. 1995 Jul-Aug;6(4):395-400
references.pmid: 7578359
references.citation: Carbohydr Res. 1992 Jun 4;230(1):63-77
references.pmid: 1324799
references.citation: Biochem J. 2008 Nov 1;415(3):333-44
references.pmid: 18842113
references.citation: J Proteome Res. 2009 Jul;8(7):3712-26
references.pmid: 19463016
references.citation: Chem Biol. 2004 Apr;11(4):535-46
references.pmid: 15123248
references.citation: Org Biomol Chem. 2010 Jan 7;8(1):66-76
references.pmid: 20024134
references.citation: Org Lett. 2004 Aug 19;6(17):2853-5
references.pmid: 15330631
references.citation: Mol Cell Proteomics. 2011 Apr;10(4):M110.003178
references.pmid: 21263009
references.citation: Biochem J. 1996 May 1;315 ( Pt 3):709-13
references.pmid: 8645147
references.citation: J Physiol. 2005 Jul 15;566(Pt 2):295-300
references.pmid: 15860522
references.citation: J Am Chem Soc. 2003 Apr 23;125(16):4686-7
references.pmid: 12696868
references.citation: Nat Methods. 2005 Sep;2(9):691-7
references.pmid: 16118640
references.citation: Science. 2001 Sep 14;293(5537):2101-5
references.pmid: 11474067
references.citation: Trends Biotechnol. 2000 Feb;18(2):64-77
references.pmid: 10652511
references.citation: Biochemistry. 1995 Dec 12;34(49):16228-34
references.pmid: 8519781
references.citation: Cell Mol Life Sci. 2008 Sep;65(18):2833-41
references.pmid: 18726176
references.citation: Br J Pharmacol. 2010 Feb;159(4):772-86
references.pmid: 20105183
references.citation: J Proteome Res. 2003 Jan-Feb;2(1):43-50
references.pmid: 12643542
references.citation: Bioorg Med Chem Lett. 2002 Mar 25;12(6):911-3
references.pmid: 11958992
references.citation: Cell. 1995 Dec 15;83(6):1037-46
references.pmid: 8521504
references.citation: Angew Chem Int Ed Engl. 2009;48(52):9879-83
references.pmid: 19943299
references.citation: Nat Rev Genet. 2006 Aug;7(8):606-19
references.pmid: 16847462
references.citation: Anal Chem. 2005 Oct 1;77(19):6218-24
references.pmid: 16194081