A Phosphorylation State-specific Antibody Recognizes Hsp27, a Novel Substrate of Protein Kinase D

• Published in: The Journal of biological chemistry 2005-04, Vol.280 (15), p.15013-15019
• Main Authors: Döppler, Heike, Storz, Peter, Li, Jing, Comb, Michael J., Toker, Alex
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Antibodies - chemistry; Biochemistry - methods; Cell Line; Enzyme-Linked Immunosorbent Assay; Gene Silencing; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - metabolism; HeLa Cells; Histone Deacetylases - chemistry; HSP27 Heat-Shock Proteins; Humans; Immunoblotting; Immunoprecipitation; Intracellular Signaling Peptides and Proteins - chemistry; Molecular Chaperones; Molecular Sequence Data; Neoplasm Proteins - chemistry; Neoplasm Proteins - metabolism; Peptides - chemistry; Phosphorylation; Protein Binding; Protein Kinase C - chemistry; Protein Kinase C - metabolism; Protein Structure, Tertiary; RNA Interference; Serine - chemistry; Signal Transduction; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.C400575200

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 Ser82 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.