Identification of Mitogen-activated Protein (MAP) Kinase-activated Protein Kinase-3, a Novel Substrate of CSBP p38 MAP Kinase

doi:10.1074/jbc.271.14.8488

Identification of Mitogen-activated Protein (MAP) Kinase-activated Protein Kinase-3, a Novel Substrate of CSBP p38 MAP Kinase (*)

From the (1)Departments of Gene Expression Sciences,
(2)Molecular Immunology,
(3)Molecular Genetics, and
(4)Cellular Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

^¶ To whom correspondence should be addressed:
Dept. of Molecular Immunology/UE0548, SmithKline Beecham Pharmaceuticals, P. O. Box 1539, King of Prussia, PA 19406-0939;
Tel.: 610-270-7691; Fax: 610-270-7962.

Abstract

CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.

Footnotes

↵^§ These two authors contributed equally to this work and should both be considered as co-first authors.
↵* 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(™)/EMBL Data Bank with accession number(s) U43784.
↵¹ The abbreviations used are:

MAP

mitogen-activated protein

AD

activation domain

BD

binding domain

CSBP

CSAID(™)-binding protein

GST

glutathione S-transferase

HA

hemagglutinin

HSP

heat shock protein

JNK

c-Jun N-terminal kinase

MAPKAP kinase

mitogen-activated protein kinase-activated protein kinase

PMA

phorbol 12-myristate 13-acetate

PAGE

polyacrylamide gel electrophoresis

TNF

tumor necrosis factor

ERK

extracellular signal-regulated kinase

kb

kilobase(s).
↵²E. Rheaume, personal communication.
↵³S. Kumar, unpublished studies.
- Received January 6, 1996.
- Revision received February 14, 1996.