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First published online 25 August 2004
doi: 10.1242/jcs.01333

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Cdk5 and Trio modulate endocrine cell exocytosis

¹ Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA
² Department of Anatomy, Institute of Biomedicine, University of Helsinki, Haartmaninkatu 8, Biomedicum, FI-00014 Helsinki, Finland

View larger version (37K): [in a new window]   Fig. 1. Cdk5 plays a role in exocytosis in pituitary endocrine cells. Cultured rat anterior pituitary cells were pre-incubated with (+Ros) or without (-Ros) roscovitine (10 µM) for 4 hours and then incubated with the same medium for two collection periods of 30 minutes each (B1 and B2) followed by incubation in medium containing secretagog (2 mM BaCl2, ST) with or without drug for 30 minutes. The media were collected and assayed for peptidylglycine -hydroxylating monooxygenase activity (PHM) (A). Extracts of rat anterior pituitary (AP), neurointermediate lobe (NIL), superior cervical ganglion (SCG) and cerebral cortex (CTX) were probed for Cdk5 (B, top) and p35 (B, bottom) by western blot. The same tissues were tested for p39 expression by RT-PCR (C, top). ß-actin amplification was carried out in parallel to provide a loading control (C, bottom). One representative experiment (of at least three) is shown.

View larger version (39K): [in a new window]   Fig. 2. Cdk5 plays a role in exocytosis of multiple pituitary hormones. Media from cultured rat anterior pituitary cells treated with or without roscovitine (as in Fig.1A) were analyzed for prolactin (PRL) (A) or growth hormone (GH) (B) by western blot and for ACTH (C) by radioimmunoassay after a basal-basal-stimulation regimen as in Fig. 1A. One representative experiment (of at least three) is shown. (D) Immunocytochemical analysis was performed by co-staining fixed cultures for prolactin and Cdk5 (upper) or GH and Cdk5 (lower); bar, 10 µm.

View larger version (81K): [in a new window]   Fig. 3. Roscovitine causes cortical actin rearrangement. Anterior pituitary cells maintained in control medium (-Ros) or treated with 10 µM roscovitine (+Ros) were fixed and visualized simultaneously for F-actin using TRITC-phalloidin (top) and prolactin (center) (A) or F-actin (top) and growth hormone (center) (B). Bottom, merged images; bar, 10 µm.

View larger version (80K): [in a new window]   Fig. 4. Treatment with roscovitine alters granule localization beneath the plasma membrane. (A) Representative somatotropes from cultures treated with roscovitine as in Fig. 3 (- or +Ros) and then prepared for electron microscopy. Asterisks mark granules closely apposed to the plasma membrane; arrowheads mark granules separated from the plasma membrane; bar, 500 nm.(B) Granules were pooled based on their distance from the plasma membrane (top left) or from other granules (top right) in somatotropes treated with (filled bars) or without roscovitine (empty bars). Kolmogorov-Smirnov cumulative distributions for granule-to-membrane (bottom left) and granule-to-granule (bottom right) distances are shown. (C) Morphological identification of somatotropes in electron micrographs was confirmed by immuno-gold labeling of growth hormone; bar, 500 nm.

View larger version (37K): [in a new window]   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.

View larger version (21K): [in a new window]   Fig. 6. Roscovitine inhibits Trio-GEF activity. (A) hEK293 cells stably expressing PAM-1 were transiently transfected with vector, myc-tagged Trio (Trio) or Myc-tagged Trio-GEF1 (T-GEF1). Control cells (-) and cells pre-treated with roscovitine (+) were extracted and aliquots were probed for Myc-tagged products (top) and total Rac (center); activated Rac was assessed following adsorption to GST-Pak-CRIB bound to glutathione agarose (bottom). A representative experiment is shown. (B) Rac activation was quantified from three independent assays; error bars show standard deviation.

View larger version (37K): [in a new window]   Fig. 7. Roscovitine reduces the fraction of Rac associated with secretory granules and the plasma membrane. (A) Proportional aliquots of the crude nuclear (P1), secretory granule/plasma membrane enriched (P2), microsomal (P3) and cytosol (SN) fractions of cultured pituitary cells treated or not with roscovitine (+ or -Ros) were analyzed by immunoblotting for Rac (top) and actin (bottom); similar results were observed in three experiments. (B) Cultured anterior pituitary cells were either treated with roscovitine (+Ros) or not (-Ros), fixed and stained with a Rac antibody and an ACTH antibody. Arrowheads indicate Rac staining under the plasma membrane; bars, 5 µm.

View larger version (33K): [in a new window]   Fig. 8. A Role for Cdk5 and Trio in regulated secretion from pituitary endocrine cells. Cdk5 and p39 are highly expressed in the pituitary. Inhibition of Cdk5 by roscovitine results in a decrease in the ability of BaCl2 to stimulate hormone release, in the accumulation of subplasma membrane filamentous actin and in a decrease in the ability of cortical large dense core vesicles to approach the plasma membrane or each other. Trio, a substrate for Cdk5 and Rho GEF, interacts with the cytosolic domain of PAM, a membrane protein localized to the regulated secretory pathway in pituitary endocrine cells. The ability of Trio to activate Rac is decreased following roscovitine treatment. Phosphorylation of Trio by Cdk5/p39 and local activation of Rac may play a role in coordinating the complex changes in the organization of the subplasma membrane actin cytoskeleton that must accompany regulated secretion and subsequent endocytosis (Bokoch, 2003).