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Activation of protein kinase C triggers cortical granule exocytosis in Xenopus oocytes

Department of Molecular & Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA 90095, USA

View larger version (57K): [in a new window]   Fig. 2. Subcellular distribution of cortical granule lectin immunoreactivity before and after PMA-induced secretion. Single stage VI oocytes were incubated without or with PMA (1 µM) for 30 minutes as indicated. After recovering the fluid surrounding each oocyte (Rel), the oocytes were fractionated (see Materials and Methods) to generate supernatant (Sup) and pellet (Pel) fractions for immunoblot analysis using cgl antibody. Samples were not reduced with DTT (see Fig. 1). The +PMA example shown here reflects the most extensive release of cgl immunoreactivity observed in six trials.

View larger version (42K): [in a new window]   Fig. 1. PMA-induced secretion of protein and immunoreactive cortical granule lectin from Xenopus oocytes. Groups of 75 stage II or single stage VI oocytes were incubated without or with PMA (1 µM) as indicated. After 30 minutes, the fluid surrounding the oocytes was collected for analysis by SDS-polyacrylamide gel and Coomassie staining or by western blot using antibodies against cgl. Samples were treated with DTT, as indicated. Mobility of molecular weight standards is indicated in kDa.

View larger version (37K): [in a new window]   Fig. 3. Concentration-response study of PMA-induced secretion of cortical granule lectin. Single oocytes were incubated in increasing concentrations of PMA, as indicated. After 1 hour, the fluid surrounding each oocyte was collected for analysis by SDS-polyacrylamide gel and Coomassie staining. The results show the smear of cgl between 35 and 50 kDa (see also Fig. 1) for two separate sets of oocytes. Pilot experiments revealed that secretion of cgl induced by 10 nM PMA was complete within 1 hour.

View larger version (43K): [in a new window]   Fig. 4. Effect of Gö6976 (A) and Gö6983 (B) on PMA-induced secretion of cortical granule lectin. Oocytes were preincubated (10-15 minutes) with the indicated concentration of drug and then incubated for 1 hour with the addition of PMA (20 nM). Released cgl was detected as in Fig. 3.

View larger version (28K): [in a new window]   Fig. 5. Detection of PKc isoform immunoreactivity in oocyte fractions with or without exposure to PMA. (A) Single stage VI oocytes were fractionated into supernatant (Sup) and pellet (Pel) fractions as in Fig. 2 before (-PMA) or after treatment with PMA (1 µM for 30 minutes, +PMA). These fractions were tested for the presence of immunoreactivity for PKC isoforms using isoform-specific antibodies. The approximate mass (in kDa) of each isoform was: delta (75); epsilon (80); eta (76); mu (105); theta (79). Preadsorption of the primary antibody with peptide immunogen abolished these bands. (B) PKC immunoreactivity in Xenopus oocytes and the brain and effect of antibody preadsorption. The results show the immunoreactive bands that are detected in supernatant (sup) and pellet (pel) fractions of oocytes probed with PKC antibody with or without preadsorption of the antibody with peptide immunogen. Immunoreactivity in a sample of extract (approximately 25 µg protein) from Xenopus brain is shown as a control.

View larger version (22K): [in a new window]   Fig. 6. Effect of over-expressing various PKC constructs on cortical granule lectin secretion from oocytes. Individual stage VI oocytes were injected with cRNA encoding wild-type (WT) or constitutively active (CA) PKC eta or delta. After incubation for 20 hours, the fluid surrounding each oocyte was collected for detection of released cgl (A). The oocytes were extracted for immunoblot analysis to verify the expression of PKC eta or delta (B). Under the conditions of immunoblot exposure in (B), endogenous expression of PKC eta or delta was essentially undetectable. Densitometric analysis revealed that injected oocytes expressed more than 50 times the endogenous level of each PKC isoform. Endogenous expression of these proteins is documented in Fig. 5.

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