A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D.
The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser(82) in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.
Döppler
Heike
H
Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Storz
Peter
P
Li
Jing
J
Comb
Michael J
MJ
Toker
Alex
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D020868:Gene Silencing; D055551:HSP27 Heat-Shock Proteins; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006655:Histone Deacetylases / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection
D000906:Antibodies; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D010455:Peptides; C092117:RIN1 protein, human; D012694:Serine; C089212:protein kinase D; D011493:Protein Kinase C; C404500:HDAC5 protein, human; D006655:Histone Deacetylases