Autophosphorylation of grp94 (endoplasmin)

P. Csermely, Y. Miyata, T. Schnaider, I. Yahara

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

The 94-kDa glucose-regulated protein (endoplasmin, grp94) is an abundant member of the 90-kDa molecular chaperons 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 κ(m) values of 243 ± 14 μM and 116 ± 23 μM, 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.

Original languageEnglish
Pages (from-to)6381-6388
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number11
DOIs
Publication statusPublished - 1995

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Adenosine Triphosphate
Extreme Heat
Binding Sites
Proteolysis
HSP90 Heat-Shock Proteins
Affinity chromatography
Casein Kinase II
Phosphorylation
Molecular Chaperones
Papain
Chymotrypsin
Threonine
Protein Kinase Inhibitors
Concanavalin A
Heat-Shock Proteins
Guanosine Triphosphate
Affinity Chromatography
Endoplasmic Reticulum
Serine
Sequence Analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Autophosphorylation of grp94 (endoplasmin). / Csermely, P.; Miyata, Y.; Schnaider, T.; Yahara, I.

In: Journal of Biological Chemistry, Vol. 270, No. 11, 1995, p. 6381-6388.

Research output: Contribution to journalArticle

Csermely, P, Miyata, Y, Schnaider, T & Yahara, I 1995, 'Autophosphorylation of grp94 (endoplasmin)', Journal of Biological Chemistry, vol. 270, no. 11, pp. 6381-6388. https://doi.org/10.1074/jbc.270.11.6381
Csermely, P. ; Miyata, Y. ; Schnaider, T. ; Yahara, I. / Autophosphorylation of grp94 (endoplasmin). In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 11. pp. 6381-6388.
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AB - The 94-kDa glucose-regulated protein (endoplasmin, grp94) is an abundant member of the 90-kDa molecular chaperons 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 κ(m) values of 243 ± 14 μM and 116 ± 23 μM, 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.

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