Molecular cloning of a new member of the p21-Cdc42/Rac-activated kinase (PAK) family.
A number of "target" proteins for the Rho family of small GTP-binding proteins have now been identified, including the protein kinases ACK and p65PAK (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). The purified serine/threonine kinase p65PAK has been shown to be directly activated by GTP-Rac1 or GTP-Cdc42. Here we report the cDNA sequence encoding a new brain-enriched PAK isoform beta-PAK, which shares 79% amino acid identity with the previously described alpha-isoform. Their mRNAs are differentially expressed in the brain, with alpha-PAK mRNA being particularly abundant in motor-associated regions. In vitro translation products of the alpha- and beta-PAK cDNAs exhibited relative molecular masses of 68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A specific beta-PAK peptide sequence was obtained from rat brain-purified p65PAK. Recombinant alpha- and beta-PAKs exhibited an increase in kinase activity mediated by GTP-p21 induced autophosphorylation. Cdc42 was a more potent activator in vitro of alpha-PAK kinase, and the fully activated enzyme is 300 times more active than the unphosphorylated form. Interestingly the down-regulation in the binding of p21s to recombinant beta-PAK and brain p65PAK, which is observed upon kinase activation does not occur with recombinant alpha-PAK.
Manser
E
E
Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore.
Chong
C
C
Zhao
Z S
ZS
Leung
T
T
Michael
G
G
Hall
C
C
Lim
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D018797:Cell Cycle Proteins / Q000502:physiology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins