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Fig. 5. Trio, a RhoGEF that interacts with a secretory pathway membrane protein, is highly expressed in anterior pituitary and is a Cdk5 substrate. (A) Data obtained from a yeast two-hybrid screen of a pituitary cDNA library, using the cytosolic domain of PAM as bait, are summarized. The products of PAM processing and their membrane topology are shown. Kalirin, a paralog of Trio, was previously identified as a PAM interactor. The region of rat Trio identified is identical in amino acid sequence to human Trio, residues 695-1136. The predicted structures of Kalirin and Trio, and the region of each that interacts with PAM, are shown. Consensus sites for Cdk5 phosphorylation are indicated for Trio. (B) Extracts of an AtT-20 cell line stably expressing PAM-1 were incubated with glutathione-Sepharose beads that had been incubated with GST-Trio-spectrin fusion protein (Trio), GST or buffer (GT). Adsorbed proteins were eluted and analyzed by immunoblotting using an antibody specific for PAM; the amount of input was 10% of that of bound fractions. (C) Extracts (20 µg protein) of rat anterior pituitary (AP), neurointermediate lobe (NIL), superior cervical ganglion (SCG) and cerebral cortex (Ctx) were probed with a Trio antibody; extracts of cells transiently expressing Trio or Kalirin-12 (K12) were analyzed at the same time. (D) AtT-20 cells transiently expressing myc-tagged Trio were visualized with antibody to the myc epitope; bar, 10 µm. (E) Three peptides encompassing the potential Cdk5 phosphorylation sites in Trio (shown in A) were synthesized; the essential Pro residue in the first site was mutated to Ala, yielding a control peptide, SAVR. The peptides were incubated for 30 minutes with immunoprecipitated p35/Cdk5 and 32P-
-ATP in kinase assays in the absence (white bars) or presence (black bars) of 10 µM roscovitine. Three independent experiments were performed.
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