Autophosphorylation of grp94 (endoplasmin).
The 94-kDa glucose-regulated protein (endoplasmin, grp94) is an abundant member of the 90-kDa molecular chaperone family in the endoplasmic reticulum. We have found earlier that the 50% homologous 90-kDa heat shock protein, hsp90, has ATP-binding site(s) and autophosphorylating activity (Csermely, P., and Kahn, C. R. (1991) J. Biol. Chem. 266, 4943-4950). In the present paper we demonstrate that highly purified grp94 is also able to autophosphorylate itself on serine and threonine residues. grp94 can be freed from the co-purifying casein kinase II by concanavalin A affinity chromatography, and its phosphorylation is unaffected by activators and inhibitors of numerous protein kinases known to associate with the homologous hsp90. The autophosphorylation persists in immunoprecipitates and in SDS-polyacrylamide gel-purified and renatured grp94. Autophosphorylation displays a monomolecular kinetics, is activated by micromolar calcium concentrations, has an extreme heat stability, and can utilize both ATP and GTP with relatively high km values of 243 +/- 14 microM and 116 +/- 23 microM, respectively. Sequence analysis of grp94 shows the presence of two ATP-binding sites. The major product of limited proteolysis of grp94 by chymotrypsin or papain is an N-terminal 85-kDa fragment that can bind to ATP-agarose but does not show autophosphorylation. Our data suggest that grp94 has an enzymatic function analogous in many respects to the similar activity of hsp70, hsp90, and grp78 (BiP). Autophosphorylation may participate in/regulate the complex formation of these proteins, so it may be involved in their chaperone function.
Csermely
P
P
Institute of Biochemistry I, Semmelweis University, School of Medicine, Budapest, Hungary.
Miyata
Y
Y
Schnaider
T
T
Yahara
I
I
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D016384:Consensus Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum; D006160:Guanosine Triphosphate / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides; D009994:Osmolar Concentration; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002413:Cations, Divalent; D018841:HSP90 Heat-Shock Proteins; D008562:Membrane Glycoproteins; D009842:Oligopeptides; C053387:endoplasmin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases