PKC- and Src-mediated phosphorylation of GRIP1 heightens GRASP-1 - GRIP1 coupling in prenatal cocaine-exposed P21 rats.

<p>(<b>a</b>) Immunoaffinity-purified native GRIP1 from frontal cortices of either prenatal saline (<b>S</b>) - or cocaine (<b>C</b>) -exposed rats was treated with vehicle or 100 µg/ml of alkaline phosphatase. Following completion of dephosphorylation by addition of inhibitors, phosphate-free GRIP1 was phosphorylated by recombinant PKC or Src in the presence of ATP. The reaction was terminated by specific PKC or Src inhibitors and GRIP1 was analyzed for phospho-serine and -tyrosine by Western blotting. The interaction between GRASP-1 and GRIP1 with different phosphorylation states was assessed <i>in vitro</i> by incubation of purified GRASP-1 derived from either prenatal saline- or cocaine-exposed rats with purified, phosphorylated GRIP1. GRASP-1 - GRIP1 complexes were isolated along with GRIP1 by immunoprecipitation and levels of GRASP-1 associated with GRIP1 were determined by Western blotting. (<b>b</b>) Densitometric quantification of the top blot shown in <b>a</b>. <i>n</i> = 4 for each group. Data are means ± s.e.m. of the optical intensity. The statistical significance was evaluated by Newman-Keuls multiple comparisons that followed one-way ANOVA. *p<0.01 compared to native GRIP1 from saline-treated group. #p<0.01 compared with dephosphorylated GRIP from both saline- and cocaine-treated groups. +p<0.01 compared with native GRIP1 from cocaine-treated group.