CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

Abstract

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.

Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin

“…Next, to investigate whether other phosphatases might affect acinar cell responses, the effects of inhibiting the non-Ca 2ϩ -dependent serine/threonine phosphatases PP1 and PP2A using okadaic acid were examined (36). In acinar cells, 10 nM okadaic acid has been used to inhibit PP1 and PP2A activity without affecting calcineurin (PP2B) (35). Okadaic acid (10 nM) pretreatment did not inhibit caerulein-induced chymotrypsin acti- Fig.…”

CARHSP1 Is Required for Effective Tumor Necrosis Factor Alpha mRNA Stabilization and Localizes to Processing Bodies and Exosomes

“…CARHSP1 contains a cold-shock domain with two RNA binding motifs (20,23); cold-shock domains preferentially bind polypyrimidine regions of singlestranded RNA and DNA and regulate ribosomal translation, mRNA degradation, and the rate of transcription termination. To date, there is limited information regarding CARHSP1, with the published literature focusing primarily on the signaling pathways that phosphorylate and dephosphorylate CARHSP1 (2,13,17,23,27). These reports demonstrate that CARHSP1 is phosphorylated through the p85 phosphatidyl inositol 3-kinase (PI3K)-Akt signaling axis on Ser-53 (23).…”

“…To date, there is limited information regarding CARHSP1, with the published literature focusing primarily on the signaling pathways that phosphorylate and dephosphorylate CARHSP1 (2,13,17,23,27). These reports demonstrate that CARHSP1 is phosphorylated through the p85 phosphatidyl inositol 3-kinase (PI3K)-Akt signaling axis on Ser-53 (23). The kinase DYRK2 has also been shown to phosphorylate CARHSP1 on Ser-30, -32, and -42 in vitro (27).…”

Inhibition of Gluconeogenic Genes by Calcium-regulated Heat-stable Protein 1 via Repression of Peroxisome Proliferator-activated Receptor α

“…To our knowledge, this may be due to increased protein stability and increased translation of CAR-HSP1 protein after refeeding. As a phosphoprotein, CARHSP1 is phosphorylated at multiple sites (7)(8)(9). Our data suggest that the 31-65 amino acid sequence located in the N-terminal domain, which is rich in phosphorylation sites, is necessary for CARHSP1 to inhibit PPAR␣ activity (Fig.…”

“…CARHSP1 is serinephosphorylated by Akt, SGK1 (serum-and glucocorticoid-induced protein kinase 1) and RSK (p90 ribosomal S6 kinase) in response to growth factors such as EGF and insulin-like growth factor-1 (IGF-1) (7). On the other hand, CARHSP1 can be dephosphorylated by Ca 2ϩ /calmodulin-regulated protein phosphatase calcineurin (PP2B) (8). Coordinated phosphorylation and dephosphorylation of CARHSP1 contribute to the multiple phosphorylated isoforms of CARHSP1 in acinar cells (9).…”