Ge. Benner et al., ACTIVATION OF AN S6 H4 KINASE (PAK-65) FROM HUMAN PLACENTA BY INTRAMOLECULAR AND INTERMOLECULAR AUTOPHOSPHORYLATION/, The Journal of biological chemistry, 270(36), 1995, pp. 21121-21128
The S6/H4 kinase purified from human placenta catalyzes phosphorylatio
n of the S6 ribosomal protein, histone H4, and myelin basic protein. I
n vitro activation of the p60 S6/H4 kinase requires removal of an auto
inhibitory 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 SVIDP
VPAPVGD-SHVDGAAK (site 2). These sequences identify S6/H4 kinase as th
e rac-activated PAK65 (Martin, G. A., Bollag, G., McCormick, F. and Ab
o, A. (1995) EMBO J. 14, 1971-1978). Site 1 phosphorylation is most ra
pid, but activation does not occur until site 2 is autophosphorylated.
The site 1 phosphorylation occurs by an intramolecular mechanism wher
eas site 2 autophosphorylation occurs by an intermolecular mechanism.
A model is proposed in which phosphorylation of sites 1 and 2 occurs s
equentially. The model proposes that trypsin treatment of the inactive
holoenzyme removes an inhibitory rac-binding domain which blocks MgAT
P access to the catalytic site. The pseudosubstrate domain at site 1 i
s autophosphorylated and subsequent bimolecular autophosphorylation at
site 2 fully opens the catalytic site. Phosphorylation by a regulator
y protein kinase may occur at site 2 in vivo.