Purification and characterization of a novel physiological substrate for calcineurin in mammalian cells.
Although the calcium/calmodulin-regulated protein phosphatase calcineurin has been shown to play a role in a number of intracellular processes, relatively few of the downstream phosphoproteins that are dephosphorylated by this enzyme in cells have been described. Calcineurin was previously shown to play a role in amylase secretion by rat pancreatic acinar cells and to specifically dephosphorylate a 24-kDa cytosolic protein. The present study describes the purification and characterization of this novel phosphoprotein, termed CRHSP-24 (calcium-regulated heat-stable protein with a molecular mass of 24 kDa). Microgram quantities of CRHSP-24 were purified from a large-scale rat pancreas preparation in a procedure involving heat and acid precipitation, anion-exchange chromatography, preparative electrophoresis, electroelution, and two-dimensional electrophoresis. Internal amino acid sequence was obtained from two peptides following trypsin digestion and high pressure liquid chromatography. Both sequences matched with 100% identity nucleotide sequences of expressed sequence tags from human placenta and rat PC-12 cells. Two CRHSP-24 transcripts of 0.7 and 2. 9 kilobases were detected in multiple rat tissues by Northern analysis, whereas a single 24-kDa protein was observed by Western blotting. The CRHSP-24 protein is 147 amino acids in length, is composed of nearly 14% proline, and is phosphorylated entirely on serine residues. Western analysis and 32P metabolic labeling of acini revealed CRHSP-24 to be maximally phosphorylated in control cells and to undergo a rapid sustained dephosphorylation on at least 3 serine residues in response to calcium-mobilizing stimuli. Dephosphorylation of CRHSP-24 was completely inhibited by pretreatment of acini with cyclosporin A or FK506. Furthermore, the inhibitory effects of FK506 were blocked by excess rapamycin. The ubiquitous expression of CRHSP-24 in rat tissues suggests that this novel calcineurin substrate plays a common role in calcium-mediated signal transduction.
Groblewski
G E
GE
Department of Physiology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109-0622, USA.
Yoshida
M
M
Bragado
M J
MJ
Ernst
S A
SA
Leykam
J
J
Williams
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D004247:DNA; D004268:DNA-Binding Proteins; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D007106:Immune Sera; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000494:pharmacology; D016716:PC12 Cells; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors
C114342:CARHSP1 protein, human; D065095:Calcineurin Inhibitors; C513278:Carhsp1 protein, rat; D004268:DNA-Binding Proteins; D007106:Immune Sera; D007166:Immunosuppressive Agents; D010750:Phosphoproteins; D014157:Transcription Factors; D019319:Okadaic Acid; D012694:Serine; D004247:DNA; D004492:Edetic Acid; D019703:Calcineurin