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. 2006 Aug;17(8):3508–3520. doi: 10.1091/mbc.E05-12-1158

Figure 5.

Figure 5.

ROCK directly phosphorylates FAK on Ser732 in response to VEGF. (A) BAECs transiently expressing EGFP, wild-type myc-tagged-ROCK I or constitutively active Myc-tagged ROCK I-Δ3 were treated or not with 10 ng/ml VEGF for 5 min and extracted. The Myc-tagged ROCK protein was immunoprecipitated using anti-Myc mouse antibody. Myc-tagged ROCK protein was processed for an immunocomplex in vitro kinase assay using MLC as substrate in the presence of 0.5 mM ATP during 20 min. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was processed for immunodetection of phospho-Ser19 within MLC (top) and Myc-tagged ROCKs (middle). Ponceau Red staining of total MLC is shown as internal control for the amount of added substrate. (B) BAECs were processed as in described A, except that FRNK was used as substrate. The membrane was processed for immunodetection of phospho-Ser732 within FAK (top). Myc-tagged ROCKs are shown in the middle panel, and Ponceau Red staining to monitor FRNK in the bottom panel. Representative blots are shown, and quantification of the blots from duplicate samples from five separate experiments (means ± SD) is shown. (C and D) Direct in vitro kinase assay was performed using increasing amounts of constitutively active form of ROCK I that was incubated with FRNK (C) or MLC (D) as substrates. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was processed for immunodetection of phospho-Ser732 within FRNK or phospho-Ser19 within MLC. Immunodetection of total FRNK and total MLC are shown (bottom). (E) Quiescent HUVECs were electroporated with 40 pmol of siRNA that specifically targets GAPDH mRNA or with increasing concentrations of siRNAs that specifically target ROCK I mRNA (smart pools from Dharmacon). Forty-eight hours later, cells were treated for 10 min with 5 ng/ml VEGF, and proteins were extracted separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was processed for immunodetection of phospho Ser732 FAK, ROCK I, total FAK, or GAPDH.