The Mixed Lineage Kinase SPRK Phosphorylates and Activates the Stress-activated Protein Kinase Activator, SEK-1

doi:10.1074/jbc.271.32.19025

The Mixed Lineage Kinase SPRK Phosphorylates and Activates the Stress-activated Protein Kinase Activator, SEK-1*

From the ^‡ Diabetes Unit and Medical Service, Massachusetts General Hospital, Boston, Massachusetts 02129 and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, the
^¶ Department of Physiology, Michigan State University, East Lansing, Michigan 48824, the
^∥ Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080, the
^” Department of Molecular Biology, Yokohama City University School of Medicine, 3-9 Fuku-ura Kanazawa-ku, Yokohama 236, Japan, and the
^‴ Howard Hughes Medical Institute and Division of Hematology/Oncology, Children's Hospital and Dana Farber Cancer Institute, Boston, Massachusetts 02115

^§ To whom correspondence should be addressed:
Diabetes Unit, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129.
Tel.: 617-726-6909; Fax: 617-726-5649; E-mail: rana{at}helix.mgh.harvard.edu

Abstract

SPRK (also called PTK-1 and MLK-3), a member of the mixed lineage kinase subfamily of (Ser/Thr) protein kinases, encodes an amino-terminal SH₃ domain followed by a kinase catalytic domain, two leucine zippers interrupted by a short spacer, a Rac/Cdc42 binding domain, and a long carboxyl-terminal proline-rich region. We report herein that SPRK activates the stress-activated protein kinases (SAPKs) but not ERK-1 during transient expression in COS cells; the p38 kinase is activated modestly (1.3-2 fold) but consistently. SPRK also activates cotransfected SEK-1/MKK-4, a dual specificity kinase which phosphorylates and activates SAPK. Reciprocally, expression of mutant, inactive SEK-1 inhibits completely the basal and SPRK-activated SAPK activity. Immunoprecipitated recombinant SPRK is able to phosphorylate and activate recombinant SEK-1 in vitro to an extent comparable to that achieved by MEK kinase-1. These results identify SPRK as a candidate upstream activator of the stress-activated protein kinases, acting through the phosphorylation and activation of SEK-1.

Footnotes

↵* This work was supported in part by grants from the National Institutes of Health and the United States Army Breast Cancer Research Program. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SAPK

stress-activated protein kinase

MAPK

mitogen-activated protein kinase

MEK

MAPK kinase

MLK

mixed lineage kinase

HA

hemagglutinin

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis.
- Received February 23, 1996.
- Revision received June 18, 1996.