Identification of c-Src tyrosine kinase substrates in platelet-derived growth factor receptor signaling

Amanchy, Ramars; Zhong, Jun; Hong, Rosa; Kim, James H.; Gucek, Marjan; Cole, Robert N.; Molina, Henrik; Pandey, Akhilesh

doi:10.1016/j.molonc.2009.07.001

« Previous Next »Molecular Oncology
Volume 3, Issue 5 , Pages 439-450, December 2009

Identification of c-Src tyrosine kinase substrates in platelet-derived growth factor receptor signaling

Ramars Amanchy
- McKusick-Nathans Institute of Genetic Medicine, Departments of Biological Chemistry, Oncology and Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
Jun Zhong
- McKusick-Nathans Institute of Genetic Medicine, Departments of Biological Chemistry, Oncology and Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
Rosa Hong
- McKusick-Nathans Institute of Genetic Medicine, Departments of Biological Chemistry, Oncology and Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
James H. Kim
- McKusick-Nathans Institute of Genetic Medicine, Departments of Biological Chemistry, Oncology and Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
Marjan Gucek
- Institute of Basic Biomedical Sciences, Mass Spectrometry/Proteomics Facility, Johns Hopkins University, Baltimore, MD 21205, USA
Robert N. Cole
- Institute of Basic Biomedical Sciences, Mass Spectrometry/Proteomics Facility, Johns Hopkins University, Baltimore, MD 21205, USA
Henrik Molina
- McKusick-Nathans Institute of Genetic Medicine, Departments of Biological Chemistry, Oncology and Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
Akhilesh Pandey
- McKusick-Nathans Institute of Genetic Medicine, Departments of Biological Chemistry, Oncology and Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
- Corresponding author. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, 733 N. Broadway, BRB 527, Baltimore, MD 21205, USA. Tel.: +1 410 502 6662; fax: +1 410 502 7544.

Received 2 May 2009; received in revised form 17 June 2009; accepted 4 July 2009. published online 17 July 2009.

Abstract

c-Src non-receptor tyrosine kinase is an important component of the platelet-derived growth factor (PDGF) receptor signaling pathway. c-Src has been shown to mediate the mitogenic response to PDGF in fibroblasts. However, the exact components of PDGF receptor signaling pathway mediated by c-Src remain unclear. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to identify Src-family kinase substrates involved in PDGF signaling. Using SILAC, we were able to detect changes in tyrosine phosphorylation patterns of 43 potential c-Src kinase substrates in PDGF receptor signaling. This included 23 known c-Src kinase substrates, of which 16 proteins have known roles in PDGF signaling while the remaining 7 proteins have not previously been implicated in PDGF receptor signaling. Importantly, our analysis also led to identification of 20 novel Src-family kinase substrates, of which 5 proteins were previously reported as PDGF receptor signaling pathway intermediates while the remaining 15 proteins represent novel signaling intermediates in PDGF receptor signaling. In validation experiments, we demonstrated that PDGF indeed induced the phosphorylation of a subset of candidate Src-family kinase substrates – Calpain 2, Eps15 and Trim28 – in a c-Src-dependent fashion.

Keywords: Kinase, Mass spectrometry, Phosphoproteomics, Phosphorylation, Signal transduction, SILAC, c-Src, PDGF