Identification of calcineurin regulated phosphorylation sites on CRHSP-24

Abstract

CRHSP-24 is a prominently regulated phosphoprotein in pancreatic acinar cells where it is the major substrate for 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, 41, 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 acinar cells and provides a molecular framework to study CRHSP-24 regulation by secretagogues and growth factor-induced kinases and phosphatases in vivo.

Figure 1

A. Calcium induced dephosphorylation of CRHSP-24 is mediated by calcineurin in HEK293 cells. Cells were transfected with rat CRHSP-24 and incubated for 48 h before being stimulated for 15 min with 5 μM ionomycin to increase intracellular calcium. When indicated, cells were exposed to 1 μM cyclosporine A (CsA) prior to ionomycin treatment. After incubation, protein lysates were separated by IEF, Western blotted, and CRHSP-24 visualized by immunoprobing and ECL detection. Arrows indicate bands containing predicted number of phosphorylated residues as described in text. B. Identification of phosphorylated serine residues in CRHSP-24. Constructs containing serine to alanine substitutions of CRHSP-24 were expressed in HEK293 cells for 48 h followed by IEF and Western blotting of unstimulated cells. Ser was replaced with Ala at the following residues: Lane 1, none; Lane 2, Ser^2,3; Lane 3, Ser¹⁷; Lane 4, Ser³⁰; Lane 5, Ser³²; Lane 6, Ser⁴¹; Lane 7, Ser⁵²; Lane 8, Ser⁵⁸; Lane 9, Ser⁷³; Lane 10, Ser⁹³; Lane 11, Ser¹¹³; Lane 12, Ser¹⁴¹; Lane 13, Ser^146,147. Arrows indicate bands containing predicted number of phosphorylated residues as described in text.

Figure 2

Multiple serine deletion defines the four phosphorylation sites of CRHSP-24. HEK293 cells were transfected with various constructs coding for wild-type or Ser to Ala mutations of CRHSP-24 for 48 h followed by IEF and Western blotting of CRHSP-24. A. Lane 1, wild-type CRHSP-24; Lane 2, pcDNA vector; Lane 3, Ser⁴¹ single mutation; Lane 4, Ser32,41 double mutation; Lane 5, Ser^30,32,41 triple mutation; Lane 6, Ser^30,32,41,52 quadruple mutation B. Comparison of Ser to Ala at position 30, 32, 41, 52 (Lane 7) to bacterially expressed wild-type CRHSP-24 (Lane 8).

Figure 3

Cyclosporine A differentially inhibits dephosphorylation of CRHSP-24 on individual phosphoserine residues. CRHSP-24 was mutated to alanine at three of the four phosphorylatable Ser residues at positions 30, 32, 41 and 52, leaving only a single phosphorylatable site as indicated. Cells were transfected and after 48 h stimulated with ionomycin followed by IEF and Western blotting of lysates as indicated in Fig. 1. Each construct was studied in three 3 replicate experiments.

Figure 4

CRHSP-24, PIPPin and lower eukaryotic orthologs contain conserved protein domain structures and sites of regulated phosphorylation. Regions of amino acid similarity are boxed and shaded by content: cold-shock domain (blue), ribonucleoprotein motifs (yellow), double-stranded RNA binding domains (green) as previously described [4]. Sites of serine phosphorylation in rat CRHSP-24 are indicated (red dots), along with positions of other serines investigated in this study (asterisks). Sites of regulated phosphorylation by protein kinases (DYRK2, PKBα, RSK) and phosphatases (PP2B) are also provided. Multiple sequence alignments were generated using ClustalW algorithms (MacVector) from GenBank sequences: rat CRHSP24, NP_690003; human CRHSP24, NP_055131; human PIPPin, NP_055275; rat PIPPin, Q63430; Bracnchiostoma florida, XP_002206249; Drosophila melanogaster CG9705, NP_730197; Anopheles gambiae, XP_315660; Aedes aegypti, XP_001659860; Hypsibius dujardini, CK326662; Schistosoma japonicum, AAW27166; Acropora palmata, DR984419.