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Title: Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

Abstract

Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic {sup 32}P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.

Authors:
 [1];  [2];  [2];  [3]
  1. Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, UCLA-CURE, Digestive Diseases Research Center and Molecular Biology Institute, University of California, Los Angeles (United States). E-mail: rwaldron@mednet.ucla.edu
  2. Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute of Neuroscience and Human Behavior, Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles (United States)
  3. Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, UCLA-CURE, Digestive Diseases Research Center and Molecular Biology Institute, University of California, Los Angeles (United States)
Publication Date:
OSTI Identifier:
20991322
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 356; Journal Issue: 2; Other Information: DOI: 10.1016/j.bbrc.2007.02.142; PII: S0006-291X(07)00389-0; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CALMODULIN; ELECTROPHORESIS; FLUORIDES; GELS; GENE MUTATIONS; IN VITRO; IN VIVO; LEAD SULFIDES; MASS SPECTROSCOPY; ONCOGENES; PHOSPHATES; PHOSPHORUS 32; PHOSPHORYLATION; TRANSCRIPTION FACTORS

Citation Formats

Waldron, Richard T., Whitelegge, Julian P., Faull, Kym F., and Rozengurt, Enrique. Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.02.142.
Waldron, Richard T., Whitelegge, Julian P., Faull, Kym F., & Rozengurt, Enrique. Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D. United States. doi:10.1016/j.bbrc.2007.02.142.
Waldron, Richard T., Whitelegge, Julian P., Faull, Kym F., and Rozengurt, Enrique. Fri . "Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D". United States. doi:10.1016/j.bbrc.2007.02.142.
@article{osti_20991322,
title = {Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D},
author = {Waldron, Richard T. and Whitelegge, Julian P. and Faull, Kym F. and Rozengurt, Enrique},
abstractNote = {Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic {sup 32}P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.},
doi = {10.1016/j.bbrc.2007.02.142},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 356,
place = {United States},
year = {Fri May 04 00:00:00 EDT 2007},
month = {Fri May 04 00:00:00 EDT 2007}
}