Activation of an S6/H4 Kinase (PAK 65) from Human Placenta by Intramolecular and Intermolecular Autophosphorylation

doi:10.1074/jbc.270.36.21121

Activation of an S6/H4 Kinase (PAK 65) from Human Placenta by Intramolecular and Intermolecular Autophosphorylation (*)

From the Division of Biochemistry and Molecular Biology, Department of Biological Sciences, University of North Texas, Denton, Texas 76203-5018

** To whom correspondence should be addressed:
Dept. of Biological Sciences, Box 5218, University of North Texas, Denton, TX 76203.
Tel.: 817-565-2968; Fax: 817-565-4136.

↵^§ Present address: Institute of Infectious Diseases, Ft. Detrick, MD 21701.
↵^¶ Present address: Friedrich Miescher Institut, 4002 Basel, Switzerland.

Abstract

The S6/H4 kinase purified from human placenta catalyzes phosphorylation of the S6 ribosomal protein, histone H4, and myelin basic protein. In vitro activation of the p60 S6/H4 kinase requires removal of an autoinhibitory domain by mild trypsin digestion and autophosphorylation of the catalytic domain (p40 S6/H4 kinase). The two autophosphorylation/autoactivation sites contain the sequences SSMVGTPY (site 1) and SVIDPVPAPVGDSHVDGAAK (site 2). These sequences identify S6/H4 kinase as the rac-activated PAK65 (Martin, G. A., Bollag, G., McCormick, F. and Abo, A.(1995) EMBO J. 14, 1971-1978). Site 1 phosphorylation is most rapid, but activation does not occur until site 2 is autophosphorylated. The site 1 phosphorylation occurs by an intramolecular mechanism whereas site 2 autophosphorylation occurs by an intermolecular mechanism. A model is proposed in which phosphorylation of sites 1 and 2 occurs sequentially. The model proposes that trypsin treatment of the inactive holoenzyme removes an inhibitory rac-binding domain which blocks MgATP access to the catalytic site. The pseudosubstrate domain at site 1 is autophosphorylated and subsequent bimolecular autophosphorylation at site 2 fully opens the catalytic site. Phosphorylation by a regulatory protein kinase may occur at site 2 in vivo.

Footnotes

↵* This research was supported by National Institutes of Health Grant 32350 (to R. A. M.). 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 abbreviations used are:

S6/H4 kinase

inactive holoenzyme

p40 S6/H4 kinase

catalytic domain of the S6/H4 kinase

TLE

thin layer electrophoresis

TLC

thin layer chromatography

HPLC

high pressure liquid chromatography

PAGE

polyacrylamide gel electrophoresis

dpm

disintegrations/minute.
- Received March 30, 1995.
- Revision received June 27, 1995.