Cellular and Molecular Life Sciences

, Volume 67, Issue 22, pp 3893–3903

Recruitment of Pyk2 to SHPS-1 signaling complex is required for IGF-I-dependent mitogenic signaling in vascular smooth muscle cells

  • Xinchun Shen
  • Gang Xi
  • Yashwanth Radhakrishnan
  • David R. Clemmons
Research Article

Abstract

In vascular smooth muscle cells, IGF-I stimulates SHPS-1/SHP2/Src complex formation which is required for IGF-I-stimulated cell proliferation. Using SHP2/Src silencing and a Pyk2/Y402F mutant, we showed that Pyk2 was also recruited to the SHPS-1 complex. Pyk2 recruitment to SHPS-1 is mediated via the interaction of Pyk2 Tyr402 and the Src in response to IGF-I. Following Src/Pyk2 association, Src phosphorylates Pyk2 on Tyr881 providing a binding site for Grb2. Cells expressing Pyk2/Y881F showed decreased Grb2 recruitment to SHPS-1 and impaired Shc/Grb2 association. This change led to reduced Erk1/2 (MAP kinase) activation and cell proliferation in response to IGF-I. Our results show that, following its recruitment to the SHPS-1 signaling complex, Pyk2 localizes Grb2 in close proximity to Shc thereby facilitating Shc/Grb2 association which leads to Erk1/2 activation in response to IGF-I. Thus, Pyk2 recruitment to SHPS-1 plays an important role in regulating the IGF-I-stimulated mitogenic response.

Keywords

IGF-I SHPS-1 Pyk2 Grb2 Shc MAP kinase Cell proliferation 

Supplementary material

18_2010_411_MOESM1_ESM.tif (1.5 mb)
Supplemental Fig. 1 Effect of SHP2/5PA mutant on Src and Pyk2 phosphorylation. Quiescent SHP2/WT or SHP2/5PA expressing SMCs were stimulated with IGF-I. Twenty micrograms of cell lysate was used for detection of (A) phosphor-Src (Y419) or phosphor-Pyk2 (Y402) and the blot was stripped and reprobed with (A) anti-Src or (B) anti-Pyk2 antibody as a loading control (TIFF 1585 kb)
18_2010_411_MOESM2_ESM.tif (1.5 mb)
Supplemental Fig. 2 The effect of SHP2/Src disruption on Pyk2 association with SHP2. (A and B) Confluent cultures were serum starved overnight and incubated with or without the SHP2 cell-permeable peptide (pept. #142, 10 µg/ml) or control peptide (cont. #136) for 1 h followed by IGF-I treatment for 5 min. The cell lysates were immunoprecipitated with anti-SHP2 antibody followed by immunoblotting for (A) Pyk2 or (B) Src. For loading control, the membrane was reprobed with anti-SHP2 antibody (TIFF 1586 kb)
18_2010_411_MOESM3_ESM.tif (1.5 mb)
Supplemental Fig. 3 Effect of Pyk2 knock-down on Src phosphorylation and SHPS-1/Src association. Quiescent Pyk2 knock-down SMCs were stimulated with IGF-I. Twenty micrograms of cell lysate was used for detection of phosphor-Src (Y419). The blot was stripped and reprobed with anti-Src antibody as a loading control (Upper panel). Cell lysates from same experiment were immunoprecipitated (IP) with anti-SHPS-1 and immunoblotted (IB) for Src. To control the loading, the blot was stripped and reprobed with anti-SHPS-1 antibody (lower panel) (TIFF 1586 kb)
18_2010_411_MOESM4_ESM.tif (1 mb)
Supplemental Fig. 4 Effect of Pyk2/Y881F mutant on SHPS-1/SHC association. Quiescent Pyk2/WT or Pyk2/Y881F expressing SMCs were stimulated with IGF-I for 5 min. Cell lysates were immunoprecipitated (IP) with anti-SHC antibody and immunoblotted (IB) for SHPS-1. To control the loading, the blot was stripped and reprobed with anti-SHC antibody (TIFF 1057 kb)
18_2010_411_MOESM5_ESM.tif (1.2 mb)
Supplemental Fig. 5 SHPS-1/Grb2 association is mediated by Pyk2. Quiescent SHC/WT or SHC/3YF expressing SMCs were stimulated with IGF-I. Cell lysates were immunoprecipitated (IP) with anti-SHPS-1 antibody and immunoblotted (IB) for Pyk2 or Grb2. To control the loading, the blot was stripped and reprobed with anti-SHPS-1 antibody (TIFF 1233 kb)

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Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • Xinchun Shen
    • 1
  • Gang Xi
    • 1
  • Yashwanth Radhakrishnan
    • 1
  • David R. Clemmons
    • 1
    • 2
  1. 1.Department of MedicineSchool of Medicine, University of North CarolinaChapel HillUSA
  2. 2.Division of EndocrinologyUniversity of North Carolina at Chapel HillChapel HillUSA

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