The intracellular signaling mechanisms by which cholecystokinin (CCK) and other secretagogues regulate pancreatic acinar function are more complex than originally realized. CCK couples through heterotrimeric G proteins of the Gq family to lead to an increase in intracellular free Ca2+, which shows spatial and temporal patterns of signaling. The actions of Ca2+ are mediated in part by activation of a number of Ca2+-activated protein kinases and the protein phosphatase calcineurin. By the process of exocytosis the intracellular messengers Ca2+, diacylglycerol, and cAMP activate the release of the zymogen granule content in a manner that is poorly understood. This fusion event most likely involves SNARE and Rab proteins present on zymogen granules and cellular membrane domains. More likely related to nonsecretory aspects of cell function, CCK also activates three MAPK cascades leading to activation of ERKs, JNKs, and p38 MAPK. Although the function of these pathways is not well understood, ERKs are probably related to cell growth, and through phosphorylation of hsp27, p38 can affect the actin cytoskeleton. The PI3K (phosphatidylinositol 3-kinase)-mTOR (mammalian target of rapamycin) pathway is important for regulation of acinar cell protein synthesis because it leads to both activation of p70S6K and regulation of the availability of eIF4E in response to CCK. CCK also activates a number of tyrosyl phosphorylation events including that of p125FAK and other proteins associated with focal adhesions.

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.

Endoplasmic reticulum (ER) stress mechanisms have been found to play critical roles in a number of diseases states, such as diabetes mellitus and Alzheimer disease, but whether they are involved in acute pancreatitis is unknown. Here we show for the first time that all major ER stress sensing and signaling mechanisms are present in exocrine acini and are activated early in the arginine model of experimental acute pancreatitis. Pancreatitis was induced in rats by intraperitoneal injection of 4.0 g/kg body wt arginine. Pancreatitis severity was assessed by analysis of serum amylase, pancreatic trypsin activity, water content, and histology. ER stress-related molecules PERK, eIF2alpha, ATF6, XBP-1, BiP, CHOP, and caspase-12 were analyzed. Arginine treatment induced rapid and severe pancreatitis, as indicated by increased serum amylase, pancreatic tissue edema, and acinar cell damage within 4 h. Arginine treatment also caused an early activation of ER stress, as indicated by phosphorylation of PERK and its downstream target eIF2alpha, ATF6 translocation into the nucleus (within 1 h), and upregulation of BiP (within 4 h). XBP-1 splicing and CHOP expression were observed within 8 h. After 24 h, increased activation of the ER stress-related proapoptotic molecule caspase-12 was observed along with an increase in caspase-3 activity and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL) staining in exocrine acini. These results indicate that ER stress is an important early acinar cell event that likely contributes to the development of acute pancreatitis in the arginine model.

Dual-specificity protein-tyrosine phosphatases (dsPTPases) have been implicated in the inactivation of mitogen-activated protein kinases (MAPKs). We have identified a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, except for the substitution of Gly for Cys in the conserved dsPTPase catalytic loop (HCXXGXXR(S/ T)). cDNA subcloning and Northern blot analysis in mouse shows poly(A ؉ ) hybridization bands of 4.6, 2.4, 1.5, and 1.2 kilobases, with highest abundance in skeletal muscle, testis, and heart. Polymerase chain reaction amplification of reverse-transcribed poly(A ؉ ) RNA revealed an alternatively spliced form of STYX containing a unique carboxyl terminus. Bacterially expressed STYX is incapable of hydrolyzing Tyr(P)-containing substrates; however, mutation of Gly 120 to Cys (G120C), which structurally mimics the active site of dsPTPases, confers phosphatase activity to this molecule. STYX-G120C mutant hydrolyzes p-nitrophenyl phosphate and dephosphorylates both Tyr(P) and Thr(P) residues of peptide sequences of MAPK homologues. The kinetic parameters of dephosphorylation are similar to human dsPTPase, Vaccinia H1-related, including inhibition by vanadate. We believe this is the first example of a naturally occurring "dominant negative" phosphotyrosine/ serine/threonine-binding protein which is structurally related to dsPTPases. Dual-specificity phosphatases (dsPTPase)1 hydrolyze phosphoserine/threonine/tyrosine-containing substrates in vitro and exhibit a substrate preference in vitro and in vivo for diphosphorylated (Thr(P)/Tyr(P)) mitogen-activated protein kinase (MAPK) homologues (reviewed in Ref. 1). Most dsPTPases are localized to the nucleus (2-4), and it has been suggested that they are responsible for the nuclear dephosphorylation and inactivation of MAPKs seen in vivo (5). All dsPTPases contain the sequence, HCXXGXXR(S/T), which has been shown to correspond to the active site of PTPases (6). The essential cysteine forms a thiophosphate intermediate during dsPTPasecatalyzed dephosphorylation (7,8). Several investigators have shown that substitution of this Cys, by Ser, in the dsPTPases, abolishes hydrolytic activity in vitro (7, 9 -12) and function in vivo (10,13,14). Interestingly, transient expression of the Cys to Ser mutant prolongs MAPK activation in vivo (12), suggesting that it may compete with native phosphatases for binding to phosphorylated MAPK. Moreover, the mutant dsPTPase co-immunoprecipitates with phosphorylated MAPK (10), indicating that the catalytic cysteine is not essential for substrate binding. This conclusion is also supported by PTPase crystallographic studies showing that Cys to Ser mutants bind sulfate (15) and tyrosine-phosphorylated peptide (16) in a manner identical with native enzymes. This paper describes the identification and isolation of a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, but contains a naturally occurring Gly residue in...

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