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MH Elliott, AJ Ghalayini; Caveolin-1 in Rod Outer Segment (ROS) Membranes is Phosphorylated by an Endogenous Tyrosine Kinase . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1405.
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Purpose: We have recently identified caveolin-1 (cav-1), a putative scaffolding protein, in photoreceptor rod outer segments and demonstrated its association with the alpha subunit of transducin (IOVS, 2001, suppl. 42, S184). Cav-1 was first discovered as a substrate for the tyrosine kinase, v-Src, in transformed cells. In this report, we show that cav-1 is specifically phosphorylated on Tyr-14 by an endogenous kinase associated with rod outer segments. Methods: Intact ROS were prepared from fresh bovine retinas on continuous sucrose gradients. ROS were incubated in the presence (ATP) and absence (control) of 1mM ATP and subjected to immunoblot analysis with antibodies directed against phosphotyrosine (PY)-containing proteins (PY99), caveolin-1 (anti-Cav-1), and phosphotyrosine-14-caveolin-1 (anti-PY14-Cav-1). The presence of PY14-Cav-1 in Triton X-100-soluble and insoluble fractions was also assessed on immunoblots. Control- and ATP-incubated ROS were immunoprecipitated with PY99 and the immune complex was subjected to immunoblot analysis with anti-cav-1. Anti-cav-1 and anti-c-Src immunoprecipitates from control- and ATP-incubated ROS were analyzed for the presence of PY14-Cav-1 and Cav-1, respectively. Results: Based on immunoprecipitation with PY99 and anti-Cav-1, and immunoblot analysis with anti-PY14-Cav-1, Cav-1 is phosphorylated on Tyr-14 in ROS incubated in the presence of ATP. As Tyr-14 is phosphorylated by Src-family kinases in non-ocular systems, we examined the association between Cav-1 and c-Src in ROS by immunoprecipitation. Cav-1 coimmunoprecipitated with c-Src in both control- and ATP-incubated ROS. Conclusions: Our results indicate that Cav-1 is phosphorylated at Tyr-14 by an endogenous kinase in ROS. Based on its association with Src, this kinase is likely to be a member of the Src-family of non-receptor tyrosine kinases. Phosphotyrosine residues in Cav-1 may provide sites for interaction with Src homology region 2-, phosphotyrosine-binding domain-containing proteins, or other ROS proteins and may promote association of these proteins with ROS membranes. Support: NIH grants EY11504; EY12190; EY13674; and Research to Prevent Blindness, Inc.
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