PKNbeta interacts with the SH3 domains of Graf and a novel Graf related protein, Graf2, which are GTPase activating proteins for Rho family.
PKNbeta is a novel isoform of PKNalpha, which is one of the target protein kinases for the small GTPase Rho. By yeast two-hybrid screening of a human embryonic kidney 293 cell cDNA library with the PKNbeta linker region containing proline-rich motifs as a bait, clones encoding Graf (GAP for Rho Associated with Focal adhesion kinase) and a novel Graf-related protein, termed Graf2, were isolated. The full length of Graf2 contains a putative PH domain, a RhoGAP domain, and an SH3 domain as well as Graf. Northern and Western blot analyses demonstrated that Graf2 is expressed in several tissues, with the highest expression in skeletal muscle. Recombinant Graf2 exhibited GTPase-activating activity toward the small GTPase RhoA and Cdc42Hs, but not toward Rac1, in vitro. The SH3 domains of Graf and Graf2 purified from Escherichia coli bound directly to PKNbeta. Graf or Graf2 was co-immunoprecipitated with PKNbeta in COS-7 cells transiently transfected with Graf or Graf2 and PKNbeta expression constructs. The catalytically active form of PKNbeta phosphorylated Graf and Graf2 in vitro. The interplay of PKNbeta and the GTPase-activating proteins, Graf and Graf2, may offer a novel mechanism regulating the Rho-mediated signaling.
Shibata
H
H
Department of Biology, Faculty of Science,Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan.
Oishi
K
K
Yamagiwa
A
A
Matsumoto
M
M
Mukai
H
H
Ono
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
C486050:ARHGAP10 protein, human; C115971:ARHGAP26 protein, human; D020690:GTPase-Activating Proteins; C061570:protein kinase N; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein