Cholecystokinin (CCK) and other pancreatic secretagogues have recently been shown to activate signaling kinase cascades in pancreatic acinar cells, leading to the activation of extracellular signal-regulated kinases and Jun N-terminal kinases. We now show the presence of a third kinase cascade activating p38 mitogen-activated protein (MAP) kinase in isolated rat pancreatic acini. CCK and osmotic stress induced by sorbitol activated p38 MAP kinase within minutes; their effects were dosedependent, with maximal activation of 2.8-and 4.4-fold, respectively. The effects of carbachol and bombesin on p38 MAP kinase activity were similar to those of CCK, whereas phorbol ester, epidermal growth factor, and vasoactive intestinal polypeptide stimulated p38 MAP kinase by 2-fold or less. Both CCK and sorbitol also increased the tyrosyl phosphorylation of p38 MAP kinase. Using the specific inhibitor of p38 MAP kinase, SB 203580, we found that p38 MAP kinase activity was required for MAP kinase-activated protein kinase-2 activation in pancreatic acini. SB 203580 reduced the level of basal phosphorylation and blocked the increased phosphorylation of Hsp 27 after stimulation with either CCK or sorbitol. CCK treatment induced an initial rapid decrease in total F-actin content of acini, followed by an increase after 40 min. Preincubation with SB 203580 significantly inhibited these changes in F-actin content. Staining of the actin cytoskeleton with rhodamine-conjugated phalloidin and analysis by confocal fluorescence microscopy showed disruption of the actin cytoskeleton after 10 and 40 min of CCK stimulation. Pretreatment with SB 203580 reduced these changes. These findings demonstrate that the activation of p38 MAP kinase is involved not only in response to stress, but also in physiological signaling by gastrointestinal hormones such as CCK, where activation of G q -coupled receptors stimulates a cascade in which p38 MAP kinase activates MAP kinase-activated protein kinase-2, resulting in Hsp 27 phosphorylation. Activation of p38 MAP kinase, most likely through phosphorylation of Hsp 27, plays a role in the organization of the actin cytoskeleton in pancreatic acini.

Treatment of 3T3-L1 adipocytes with insulin (IC 50 ϳ200 pM insulin) or insulin-like growth factor-1 (IC 50 ϳ200 pM IGF-1) stimulates dephosphorylation of CCAAT/ enhancer binding protein ␣ (C/EBP␣), a transcription factor involved in preadipocyte differentiation. As assessed by immunoblot analysis of one-and two-dimensional PAGE, insulin appears to dephosphorylate one site within p30C/EBP␣ and an additional site within p42C/EBP␣. Consistent with insulin causing dephosphorylation of C/EBP␣ through activation of phosphatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase inhibitors (e.g. wortmannin) blocks insulinstimulated dephosphorylation of C/EBP␣. In the absence of insulin, wortmannin or LY294002 enhance C/EBP␣ phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphorylation of C/EBP␣ in the absence of insulin. Dephosphorylation of C/EBP␣ by insulin is partially blocked by rapamycin, consistent with a model in which activation of FKBP-rapamycin-associated protein by phosphatidylinositol 3-kinase results in dephosphorylation of C/EBP␣. The dephosphorylation of C/ EBP␣ by insulin, in conjunction with the insulindependent decline in C/EBP␣ mRNA and protein, has been hypothesized to play a role in repression of GLUT4 transcription by insulin. Consistent with this hypothesis, the decline of GLUT4 mRNA following exposure of adipocytes to insulin correlates with dephosphorylation of C/EBP␣. However, the repression of C/EBP␣ mRNA and protein levels by insulin is blocked with an inhibitor of the mitogen-activated protein kinase pathway without blocking the repression of GLUT4 mRNA, thus dissociating the regulation of C/EBP␣ and GLUT4 mRNAs by insulin. A decline in C/EBP␣ mRNA and protein may not be required to suppress GLUT4 transcription because insulin also induces expression of the dominant-negative form of C/EBP␤ (liver inhibitory protein), which blocks transactivation by C/EBP transcription factors.

The Munc-18-syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologues in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18 that has been phosphorylated by Cdk5 has a significantly reduced affinity for syntaxin 1A. Finally, it is shown that Cdk5 can also bind to syntaxin 1A and that a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18-syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism.Many of the key proteins involved in membrane targeting and synaptic vesicle neurotransmitter release have been identified, and a fundamental set of interactions has been defined and placed into a model termed the SNARE hypothesis (1-4). Essential to the SNARE hypothesis is the idea that a complex of proteins is formed from soluble cytosolic proteins and from proteins integral to the synaptic vesicle (termed v-SNAREs) and target membranes (termed t-SNAREs). The soluble proteins include the ATPase N-ethylmaleimide-sensitive fusion protein (NSF) 1 and a family of proteins necessary for membrane attachment and activation of NSF termed SNAPs (soluble NSF attachment proteins). The SNAP receptors (i.e. the SNAREs) were identified as synaptic vesicle-associated membrane protein (VAMP, also termed synaptobrevin), and the plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) (2, 5). In the SNARE hypothesis, the core of the vesicle docking interaction results from the interaction of v-SNAREs with cognate t-SNAREs via coiled-coil domains to create a protein complex that further recruits cytosolic factors ␣/␤-and ␥-SNAP and then NSF to form a 20 S complex. This assemblage of proteins is required for the donor and target membranes to come into close apposition and, upon ATP hydrolysis by NSF, become fusion-competent (4, 5). A specialization imposed on the SNARE hypothesis with regard to neurotransmitter and neurohormone rele...

Previous studies have demonstrated roles for vesicle-associated membrane protein 2 (VAMP 2) and VAMP 8 in Ca 2؉ -regulated pancreatic acinar cell secretion, however, their coordinated function in the secretory pathway has not been addressed. Here we provide evidence using immunofluorescence microscopy, cell fractionation, and SNARE protein interaction studies that acinar cells contain two distinct populations of zymogen granules (ZGs) expressing either VAMP 2 or VAMP 8. Further, VAMP 8-positive granules also contain the synaptosome-associated protein 29, whereas VAMP 2-expressing granules do not. Analysis of acinar secretion by Texas red-dextran labeling indicated that VAMP 2-positive ZGs mediate the majority of exocytotic events during constitutive secretion and also participate in Ca 2؉ -regulated exocytosis, whereas VAMP 8-positive ZGs are more largely involved in Ca 2؉ -stimulated secretion. Previously undefined functional roles for VAMP and syntaxin isoforms in acinar secretion were established by introducing truncated constructs of these proteins into permeabilized acini. VAMP 2 and VAMP 8 constructs each attenuated Ca 2؉ -stimulated exocytosis by 50%, whereas the neuronal VAMP 1 had no effects. In comparison, the plasma membrane SNAREs syntaxin 2 and syntaxin 4 each inhibited basal exocytosis, but only syntaxin 4 significantly inhibited Ca 2؉ -stimulated secretion. Syntaxin 3, which is expressed on ZGs, had no effects. Collectively, these data demonstrate that individual acinar cells express VAMP 2-and VAMP 8-specific populations of ZGs that orchestrate the constitutive and Ca 2؉ -regulated secretory pathways.The exocrine pancreas is responsible for synthesizing and secreting a variety of digestive enzymes that are essential for assimilation of the diet. Secretion occurs by exocytosis of large dense core zymogen granules (ZGs) 3 localized in the apical cytoplasm of acinar cells. Similar to endocrine and neural cells, exocytosis is highly induced following acinar stimulation by secretagogues; however, acini are unique in that a significant proportion of exocytosis also occurs by a constitutive pathway under basal conditions. Secretagogue-stimulated exocytosis is mediated by G protein-coupled receptors, which signal through phospholipase C and/or adenylate cyclase, and The process of exocytosis and other membrane fusion events is widely held to be regulated by soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) interactions in cells (3-5). SNARE proteins are classified as either Q-SNARE or R-SNARE based on conserved glutamine (Q) and arginine (R) residues positioned within their characteristic coiled-coil motifs. When brought in close opposition, Q-and R-SNAREs on each membrane form a heterotrimeric complex that provides the driving force for membrane fusion. In neurons, one R-containing coil of the complex is derived from vesicle-associated membrane protein (VAMP/synaptobrevin) present on synaptic vesicles, and one Q-containing coil is contributed by syntaxin 1 located on the pre...

Sustained smooth muscle contraction is mediated by protein kinase C (PKC) through a signal transduction cascade leading to contraction. Heat-shock protein 27 (HSP27) appears to be the link between these two major events, i.e., signal transduction and sustained smooth muscle contraction. We have investigated the involvement of HSP27 in signal transduction and HSP27 association with contractile proteins (e.g., actin, myosin, tropomyosin, and caldesmon) resulting in sustained smooth muscle contraction. We have carried out confocal microscopy to investigate the cellular reorganization and colocalization of proteins and immunoprecipitation of HSP27 with actin, myosin, tropomyosin, and caldesmon as detected by sequential immunoblotting. Our results indicate that 1) translocation of Raf-1 to the membrane when stimulated with ceramide is inhibited by vasoactive intestinal peptide (VIP), a relaxant neuropeptide; 2) PKC-α and mitogen-activated protein kinase translocate and colocalize on the membrane in response to ceramide, and PKC-α translocation is inhibited by VIP; 3) HSP27 colocalizes with actin when contraction occurs; and 4) HSP27 immunoprecipitates with actin and with the contractile proteins myosin, tropomyosin, and caldesmon. We propose a model in which HSP27 is involved in sustained smooth muscle contraction and modulates the interaction of actin, myosin, tropomyosin, and caldesmon.