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.

We have isolated the human GRAF gene (for GTPase regulator associated with the focal adhesion kinase pp125(FAK)). This gene was fused with MLL in a unique t(5;11)(q31;q23) that occurred in an infant with juvenile myelomonocytic leukemia. GRAF encodes a member of the Rho family of the GTPase-activating protein (GAP) family. On the protein level, it is 90% homologous to the recently described chicken GRAF gene that functions as a GAP of RhoA in vivo and is thus a critical component of the integrin signaling transduction pathway. The particular position of the human GRAF gene at 5q31 and the proposed antiproliferative and tumor suppressor properties of its avian homologue suggest that it also might be pathogenetically relevant for hematologic malignancies with deletions of 5q. To investigate this possibility, we sequenced 4-5 individual cDNA clones from 13 cases in which one allele of GRAF was deleted. We found point mutations within the GAP domain of the second GRAF allele in one patient. In two additional patients we found an insertion of 52 or 74 bp within the GRAF cDNA that generates a reading frame shift followed by a premature stop codon. GRAF maps outside the previously defined commonly deleted 5q31 region. Nevertheless, inactivation of both alleles in at least some cases suggests that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoeitic disorders with a del(5q).

For several decades, it has been known that mental retardation (MR) is associated with abnormalities in dendrites and dendritic spines. The recent cloning of seven genes that cause nonspecific MR when mutated provides important insights in the cellular mechanisms that result in the dendritic abnormalities associated with MR. Three of the encoded proteins, oligophrenin 1, PAK3 and alpha PIX, interact directly with Rho GTPases. Rho GTPases are key signaling proteins that integrate extracellular and intracellular signals to orchestrate coordinated changes in the actin cytoskeleton essential for directed neurite outgrowth and the regulation of synaptic connectivity. Although many details of the cell biology of Rho signaling in the CNS are still unclear, a picture is unfolding showing how mutations that alter Rho signaling result in abnormal neuronal connectivity and deficient cognitive functioning in humans. Conversely, these findings illuminate the cellular mechanisms underlying normal cognitive function.

Primary or nonspecific X-linked mental retardation (MRX) is a heterogeneous condition in which affected patients do not have any distinctive clinical or biochemical features in common apart from cognitive impairment. Although it is present in approximately 0.15-0.3% of males, most of the genetic defects associated with MRX, which may involve more than ten different genes, remain unknown. Here we report the characterization of a new gene on the long arm of the X-chromosome (position Xq12) and the identification in unrelated individuals of different mutations that are predicted to cause a loss of function. This gene is highly expressed in fetal brain and encodes a protein of relative molecular mass 91K, named oligophrenin-1, which contains a domain typical of a Rho-GTPase-activating protein (rhoGAP). By enhancing their GTPase activity, GAP proteins inactivate small Rho and Ras proteins, so inactivation of rhoGAP proteins might cause constitutive activation of their GTPase targets. Such activation is known to affect cell migration and outgrowth of axons and dendrites in vivo. Our results demonstrate an association between cognitive impairment and a defect in a signalling pathway that depends on a Ras-like GTPase.

We have isolated the human GRAF gene (for GTPase regulator associated with the focal adhesion kinase pp125(FAK)). This gene was fused with MLL in a unique t(5;11)(q31;q23) that occurred in an infant with juvenile myelomonocytic leukemia. GRAF encodes a member of the Rho family of the GTPase-activating protein (GAP) family. On the protein level, it is 90% homologous to the recently described chicken GRAF gene that functions as a GAP of RhoA in vivo and is thus a critical component of the integrin signaling transduction pathway. The particular position of the human GRAF gene at 5q31 and the proposed antiproliferative and tumor suppressor properties of its avian homologue suggest that it also might be pathogenetically relevant for hematologic malignancies with deletions of 5q. To investigate this possibility, we sequenced 4-5 individual cDNA clones from 13 cases in which one allele of GRAF was deleted. We found point mutations within the GAP domain of the second GRAF allele in one patient. In two additional patients we found an insertion of 52 or 74 bp within the GRAF cDNA that generates a reading frame shift followed by a premature stop codon. GRAF maps outside the previously defined commonly deleted 5q31 region. Nevertheless, inactivation of both alleles in at least some cases suggests that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoeitic disorders with a del(5q).