Autophosphorylation of grp94 (Endoplasmin) (*)
- From the (1)Institute of Biochemistry I, Semmelweis University, School of Medicine, H-1444 Budapest, Hungary and
- the (2)Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Hon-Komagome, Bunkyo-ku, Tokyo 113, Japan
- § To whom correspondence should be addressed: Semmelweis University, Inst. of Biochemistry I, P.O. Box 260, H-1444 Budapest, Hungary. Tel./Fax: 361-266-6550.
Abstract
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 k
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.
Footnotes
-
↵* This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan; by Nissan Science Foundation; by a grant from the Human Frontier Science Program; by a fellowship of the Tokyo Metropolitan Institute of Medical Science; by the Hungarian Academy of Sciences (OTKA-T5534); and by the Hungarian Ministry of Social Welfare (ETT-202/91, ETT-681/93). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- grp78
-
the immunoglobulin heavy chain-binding protein (BiP)
- ConA
-
concanavalin A
- DnaK
-
the hsp70 homologue of Escherichia coli
- grp94
-
94-kDa glucose-regulated protein (endoplasmin, ERp99, gp96, hsp100, hsp108)
- H-7
-
1-(5-isoquinolylsulfonyl)-2-methylpiperazine
- H-8
-
N-(2[methylamino]ethyl)-5-isoquinoline-sulfonamide
- hsp70
-
70-kDa heat shock protein and its constitutive homologue, hsc70
- hsp90
-
90-kDa heat shock protein
- PAGE
-
polyacrylamide gel electrophoresis
- MES
-
4-morpholineethanesulfonic acid.
-
↵2 P. Csermely, Y. Miyata, and I. Yahara, unpublished observations.
-
↵3 P. Csermely, T. Schnaider, and C. Soti, unpublished observations.
-
- Received September 19, 1994.
- Revision received December 15, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
