CRHSP-24 is a prominently regulated phosphoprotein in pancreatic acinar cells where it is the major substrate fur the serine/threonine protein phosphatase, calcineurin, in response to secretagogues. We now identify the four regulated sites of CRHSP-24 phosphorylation as serines 30, 32, 4 1, and 52 and show that Ser(30) and Ser(32) are directly dephosphorylated by calcineurin. Coordinate phosphorylation/dephosphorylation of these four serines explains the multiple phosphorylated isoforms of CRHSP-24 present in actual cells and provides a molecular framework to Study CRHSP-24 regulation by secretagogues and growth factor-induced kinases and phosphatases in vivo. (C) 2009 Elsevier Inc. All rights reserved.