The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling.

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton:membrane linkers. However, the molecular basis for the tumor- and metastasis- suppressing activity of merlin is unknown. We have now placed merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression.

Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with Ras in activating extracellular signal-regulated kinase ERK1.

Previous reports from our laboratory described the activation of the PCPH gene into the PCPH oncogene (mt-PCPH, reported previously as Cph) by a single point mutational deletion. As a consequence, the mt-PCPH oncoprotein is a truncated form of the normal PCPH protein. Although both proteins have ribonucleotide diphosphate-binding activity, only mt-PCPH acted synergistically with a human H-Ras oncoprotein to transform murine NIH3T3 fibroblasts. We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the purified bacterial recombinant proteins have intrinsic guanosine diphosphatase (GDPase) activity. However, expression of the Syrian hamster PCPH and mt-PCPH proteins in haploid yeast strains engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes. Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the stimulatory effect of epidermal growth factor or the activated ras or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway. However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a prolonged stimulation of the phosphorylation of ERK1 but had no effect on the phosphorylation levels of ERK2. These results represent a clear difference between the mechanisms of action of PCPH and mt-PCPH and suggest that the ability to cause a sustained activation of ERK1 may be an important determinant of the transforming activity of mt-PCPH.

Identity between the PCPH proto-oncogene and the CD39L4 (ENTPD5) ectonucleoside triphosphate diphosphohydrolase gene.

PCPH was initially defined as a proto-oncogene on the basis of its frequent detection as an activated oncogene in tumorigenic Syrian hamster embryo fibroblast cell lines converted to the neoplastic state by a single treatment with the carcinogen 3-methylcholanthrene (MC). Further studies identified the translation product of the PCPH gene as a ribonucleotide-binding protein with special affinity for ribonucleoside diphosphates. Later, we showed that the PCPH protein was homologous to the product of the yeast GDA1 gene and demonstrated that it had intrinsic guanosine diphosphatase activity, although it did not complement the disrupted phenotype when expressed in gda1 null Saccharomyces cerevisiae strains. These results indicated that the primary function of PCPH was unlikely to be related to the ribonucleotide recycling function that its yeast counterpart performs in the Golgi during the process of protein glycosylation. However, taken together, our data strongly suggested that the normal cellular function of PCPH was related to ribonucleotide metabolism. We now report that PCPH is structurally and functionally identical to the mammalian ectonucleoside triphosphate diphosphohydrolase CD39L4 (ENTPD5), recently described as a member of the lymphoid activation antigen () CD39 protein family. These results may help to establish the normal cellular function of the PCPH proto-oncogene product and its role in neoplastic development during carcinogenesis.