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

J Biol Chem. 2005 Apr 15;280(15):15013-9. doi: 10.1074/jbc.C400575200. Epub 2005 Feb 22.

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 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Antibodies / chemistry*
  • Biochemistry / methods*
  • Cell Line
  • Enzyme-Linked Immunosorbent Assay
  • Gene Silencing
  • HSP27 Heat-Shock Proteins
  • HeLa Cells
  • Heat-Shock Proteins / chemistry*
  • Heat-Shock Proteins / metabolism
  • Histone Deacetylases / chemistry
  • 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

Substances

  • Antibodies
  • HSP27 Heat-Shock Proteins
  • HSPB1 protein, human
  • Heat-Shock Proteins
  • Intracellular Signaling Peptides and Proteins
  • Molecular Chaperones
  • Neoplasm Proteins
  • Peptides
  • RIN1 protein, human
  • Serine
  • protein kinase D
  • Protein Kinase C
  • HDAC5 protein, human
  • Histone Deacetylases