Identification of a mouse p21Cdc42/Rac activated kinase.
We have isolated a novel member of the mammalian PAK (p21 activated kinase) and yeast Ste20 serine/threonine kinase family from a mouse fibroblast cDNA library, designated mPAK-3. Expression of mPAK-3 in Saccharomyces cerevisiae partially restores mating function in ste20 null cells. Like other PAKs, mPAK-3 contains a putative Cdc42Hs/Rac binding sequence and when transiently expressed in COS cells, full-length mPAK-3 binds activated (GTP gamma S (guanosine 5'-3-O-(thio-triphosphate)-bound) glutathione S-transferase (GST)-Cdc42Hs and GST-Rac1 but not GST-RhoA. As expected for a putative target molecule, mPAK-3 does not bind to an effector domain mutant of Cdc42Hs. Furthermore, activated His-tagged Cdc42Hs and His-tagged Rac stimulate mPAK-3 autophosphorylation and phosphorylation of myelin basic protein by mPAK-3 in vitro. Interestingly, the amino-terminal region of mPAK-3 contains potential SH3-binding sites and we find that mPAK-3, expressed in vitro and in vivo, shows highly specific binding to the SH3 domain of phospholipase C-gamma and at least one SH3 domain in the adapter protein Nck. These results raise the possibility of an additional level of regulation of the PAK family in vivo.
Bagrodia
S
S
Department of Pharmacology, Cornell University, Ithaca, New York 14853, USA.
Taylor
S J
SJ
Creasy
C L
CL
Chernoff
J
J
Cerione
R A
RA
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D017931:DNA Primers; D018076:DNA, Complementary; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D015723:Gene Library; D005982:Glutathione Transferase / Q000096:biosynthesis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006639:Histidine; D051379:Mice; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D016324:Sequence Tagged Sites; D014162:Transfection; D054462:p21-Activated Kinases
D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006639:Histidine; D005982:Glutathione Transferase; C515912:Pak3 protein, mouse; C515913:Pak3 protein, rat; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases