Dephosphorylation of the small heat shock protein hsp25 by calcium/calmodulin-dependent (type 2B) protein phosphatase.
The dephosphorylation of the mouse small heat shock protein hsp25 within an extract obtained from Ehrlich ascites tumor cells is inhibited by the calcium chelator EGTA and at concentrations of microcystin-LR which are characteristic for inhibition of calcium/calmodulin-dependent (2B type) protein phosphatases. Furthermore, the dephosphorylation of hsp25 in the cell-free system derived from Ehrlich ascites tumor could be increased specifically by addition of the calcium/calmodulin-dependent (2B type) protein phosphatase calcineurin. Dephosphorylation of the heat shock protein hsp25 is also obtained in an in vitro system containing phosphorylated recombinant hsp25, 1 mM Ca2+, calmodulin, and calcineurin specifying hsp25 as the direct substrate for this enzyme. The expression of two isoforms of the catalytic subunit of the mouse calcium/calmodulin-dependent (2B type) protein phosphatases in Ehrlich ascites tumor cells is demonstrated by polymerase chain reaction using specific oligonucleotide primers to the catalytic and calmodulin-binding domain, respectively. Northern blot analysis using the amplified fragments as probes shows that the mRNA of one isoform of the mouse calcium/calmodulin-dependent protein phosphatase is of medium abundance in EAT cells. These data suggest a calcium/calmodulin-dependent dephosphorylation of the small stress protein in EAT cells also in vivo. Since it is known that heat shock increases the intracellular calcium level and that thermotolerance is influenced by calcium chelators, ionophores, and anti-calmodulin drugs, the changes in the degree of hsp25 phosphorylation induced by thermal stress resulting in an altered thermoresistance could be explained at least partially by the calcium/calmodulin-dependent dephosphorylation through protein phosphatases 2B.
Gaestel
M
M
Max-Delbrück-Center for Molecular Medicine, Berlin, Federal Republic of Germany.
Benndorf
R
R
Hayess
K
K
Priemer
E
E
Engel
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002286:Carcinoma, Ehrlich Tumor; D002474:Cell-Free System; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D004277:DNA, Single-Stranded; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D002118:Calcium