Activation of Protein Kinase Cascades by Osmotic Shock

doi:10.1074/jbc.270.21.12781

Activation of Protein Kinase Cascades by Osmotic Shock (*)

From the (1) Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan

^¶ To whom correspondence should be addressed. Fax: 81-75-751-3992.

Abstract

Osmotic shock induces a variety of biochemical and physiological responses in vertebrate cells. By analyzing extracts obtained from rat 3Y1 fibroblastic cells exposed to hyper-osmolar media, we have found that mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs, also known as JNKs) are both activated in response to osmotic shock. MAPKK1 (MEK1) was also activated markedly. Furthermore, Raf-1 and MEKK were activated strikingly by the osmotic shock. Activation of Raf-1 and MEKK in response to osmotic shock was detected also in PC12 cells, in which MEKK activation by the osmotic shock was much stronger than that by epidermal growth factor. Activation of SAPKs in PC12 cells by the osmotic shock was also more marked than that by epidermal growth factor. The activated MEKK phosphorylated not only MAPKKs but also XMEK2, which is distantly related to MAPKK. Recombinant wild-type XMEK2, but not kinase-negative XMEK2, was able to phosphorylate and activate recombinant SAPKα in vitro. In addition, this activity of XMEK2 was activated by the activated MEKK. These results suggest that the MAPK cascade consisting of Raf-1, MAPKK, and MAPK and the SAPK cascade consisting of MEKK, XMEK2, and SAPK are both activated in response to osmotic shock. Finally, it was found that XMEK2 is a good substrate for SAPK.

Footnotes

↵^§ S. M., H. K., and T. M. contributed equally to this work.
↵* This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, the Toray Science Foundation, and the Mitsubishi Foundation. 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.
↵¹ The abbreviation used are:

MAPK

mitogen-activated protein kinase

MAPKK

MAPK kinase

MAPKK-K

MAPKK kinase

SAPK

stress-activated protein kinase

JNK

jun N-terminal kinase

EGF

epidermal growth factor

MBP

myelin basic protein

MAP2

microtubule-associated protein 2

PAGE

polyacrylamide gel electrophoresis.
↵²T. Moriguchi, Y. Gotoh, and E. Nishida, manuscript in preparation.
↵³H. Kawasaki, Y. Gotoh, and E. Nishida, unpublished results.
↵⁴T. Moriguchi, H. Kawasaki, S. Matsuda, Y. Gotoh, and E. Nishida, unpublished observations.