Article

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

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Journal of Biological Chemistry (impact factor: 4.77). 05/2005; 280(15):15013-9. DOI:10.1074/jbc.C400575200 pp.15013-9
Source: PubMed

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.

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Keywords

activate PKD
 
antibody anti-PKD pMOTIF
 
antibody immunoreactive
 
consensus substrate phosphorylated motif
 
degenerate phosphopeptide library
 
given protein kinase
 
human heat shock protein Hsp27
 
immunoreactive phosphoproteins
 
novel PKD substrate proteins
 
novel substrate
 
optimal phosphorylation motif
 
phosphorylation state-specific antibodies
 
PKD consensus motif
 
PKD substrates RIN1
 
protein kinase D
 
putative substrates
 
recent application
 
RNA interference validated
 
Ser/Thr protein kinases
 
Silencing PKD expression