A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D

Heike Döppler, Peter Storz, Jing Li, Michael J. Comb, Alex Toker

Research output: Contribution to journalArticle

132 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)15013-15019
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number15
DOIs
StatePublished - Apr 15 2005
Externally publishedYes

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry

Cite this