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

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.

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

“…That Tyr-491 was the specific phosphotyrosine that mediated Nox4 recruitment to SHPS-1 was shown by demonstrating that cells expressing a mutant form of Nox4 (Y491F) or a cell-permeable peptide containing a Tyr to Asp substitution at position 491, and the Nox4 flanking sequence prevented Nox4/Grb2 association as well as Nox4 recruitment to SHPS-1. To confirm this result, we utilized cells that expressing a Pyk2 Y881F mutant, because our previous study had shown that Pyk2 mediates Grb2 recruitment to SHPS-1 (19). As predicted, cells expressing this mutant had attenuated Nox4 recruitment.…”

“…4E). To further confirm that Grb2 mediated Nox4 recruitment to SHPS-1, we utilized VSMC expressing a Pyk2 Y881F mutant that we had previously shown eliminated Grb2 recruitment to SHPS-1 (19). IGF-Istimulated Nox4/SHPS-1 association was not detected in the Pyk2 Y881F mutant cells (Fig.…”

“…293FT cells (Invitrogen) were prepared for generation of virus stocks, and VSMC expressing Nox4, Nox4 Y491F, and mitochondria-localized catalase were established using procedures that were described previously (18). VSMC expressing wild type Pyk2, Pyk2 Y881F, wild type SHPS-1, and cytoplasmic domain-deleted SHPS-1, were prepared as described previously (14,19).…”

“…Immunoprecipitation was performed by incubating 0.5 mg of cell lysate protein with 1 g of each of the following antibodies: anti-Src, SHPS-1, Nox4, and HA at 4°C overnight. Double immunoprecipitation was performed using a procedure described previously (19). Briefly, the first round immunoprecipitation was performed with an anti-Nox4 antibody or a nonimmune IgG (control).…”

Insulin-Like Growth Factor (IGF) Binding Protein 2 Functions Coordinately with Receptor Protein Tyrosine Phosphatase β and the IGF-I Receptor To Regulate IGF-I-Stimulated Signaling

“…The expression vector of the shRNA template for LacZ was also used as a control. The expression plasmids encoding these shRNAs were prepared as described previously (46).…”