Phosphatidylinositol (Pl)-3 kinase is one of many enzymes stimulated by growth factors. A constitutively activated mutant, p110, that functions independently of growth factor stimulation was constructed to determine the specific responses regulated by Pl-3 kinase. The p110 protein exhibited high specific activity as a Pl-3 kinase and as a protein kinase. Expression of p110 in NIH 3T3 cells induced transcription from the fos promoter. Co-expression of dominant negative Ras blocked this response. When expressed in Xenopus laevis oocytes, p110 increased the amount of guanosine 5'-triphosphate-bound Ras, caused activation of the Ras effector Raf-1, and induced Ras-dependent oocyte maturation. These findings show that Pl-3 kinase can stimulate diverse Ras-dependent cellular processes, including oocyte maturation and fos transcription.

The protein product of the retinoblastoma (RB) gene is thought to function in a pathway that restricts cell proliferation. Recently, transforming proteins from three different classes of DNA tumor viruses have been shown to form complexes with the RB protein. Genetic studies suggest that these interactions with the RB protein are important steps in transformation by these viruses. In order to understand better the function of the RB‐viral oncoprotein complexes, we have mapped the regions of the RB protein that are necessary for these associations. Two non‐contiguous regions of RB were found to be essential for complex formation with adenovirus E1A or SV40 large T antigen. These two regions are found between amino acids 393 and 572 and 646 and 772. Interestingly, these binding sites on RB overlap with the positions of naturally occurring, inactivating mutations of the RB gene. These results strongly suggest that these viral oncoproteins are targeting a protein domain that is an important site in the normal function of the RB protein.

Activins are members of the transforming growth factor beta (TGF beta) superfamily, which comprises a growing group of dimeric proteins. TGF beta and several other members of this superfamily are known to play an important role in wound healing. However, expression of activin during wound healing has not been demonstrated so far. In this study we have analyzed the expression pattern of activin and activin receptors in normal and wounded skin. We found a large induction of activin A and a minor induction of activin B mRNA expression 1 day after skin injury and high expression levels of activin A and B were found within the first 7 days after wounding. At 13 days after injury, expression of activin A mRNA had returned to the basal level, whereas high levels of activin B persisted. In situ hybridization studies revealed expression of activin A in the granulation tissue below the wound and activin B in the hyperproliferative epithelium at the wound edge and in the migrating epithelial tongue. All known types of activin receptors as well as the activin binding protein follistatin were expressed in normal and wounded skin. However, no significant induction of receptor gene expression was seen during the repair process. The distribution of activins and activin receptors in the wound suggests multiple autocrine and paracrine activities of the ligands during wound healing. Our data provide evidence for a novel function of activin and indicate that--besides TGF beta s themselves--other members of this superfamily might also play an important role in tissue repair.

NCK, an SH2-and SH3 domain-containing protein, becomes phosphorylated and associated with tyrosine kinase receptors upon growth factor stimulation. The sequence of NCK suggests that NCK functions as a linker between receptors and a downstream signaling molecule. To determine if NCK can mediate growth factorstimulated responses, we measured the ability of NCK to activate the fos promoter. We found that in NIH 3T3 cells, NCK strongly activates this promoter. The effect of NCK on the fos promoter is enhanced by c-ras and blocked by dominant negative ras. We also found that NCK binds directly to the guanine nucleotide exchange factor SOS. This interaction is mediated by the SH3 domains of NCK. These findings suggest that NCK can regulate p21 ras -dependent gene transcription through interaction with SOS protein.NCK, a 45-kDa protein consisting of one SH2 domain and three SH3 domains, is expressed in a wide range of tissues and cell lines (20). After stimulation of cells by growth factors, NCK becomes phosphorylated on Ser, Thr, and Tyr residues and associates with tyrosine kinase receptors (5,17,21,25,28). In this way, NCK resembles other proteins that have SH2 domains and associate with receptors through interaction between SH2 domains and specific phosphotyrosines (13,19,24,29). Overexpression of NCK transforms mammalian fibroblasts, suggesting that its SH2 and SH3 domains may play important roles in cell growth regulation (5, 21). However, the mechanism of action of NCK is not understood.Recent studies of SH2-and SH3 domain-containing signaling molecules that associate with tyrosine-phosphorylated receptors have shown that signaling molecules are of two general types. The first type, typified by phospholipase C-␥, is an enzyme itself. The p85 subunit of phosphatidylinositol 3-kinase (10, 27, 34) represents a second type of receptor-associated molecule, in that it probably has no defined intrinsic enzyme activity but serves as a linker that connects an enzyme, in this case the catalytic subunit of phosphatidylinositol 3-kinase, to a receptor. Another example of the second type of linking molecule is the Sem5 gene product in Caenorhabditis elegans and its mammalian counterpart, GRB2. The SH2 domain of Sem5/ GRB2 binds to tyrosine kinase receptors (22,26,33). The SH3 region of Sem5/GRB2, like the SH3 region of the Abelson protein (6, 7, 30), binds to a proline-rich motif that is a common feature of a number of molecules implicated in growth regulation (9,16,26, 32,33).The absence of a recognizable catalytic domain of NCK suggests that NCK serves as a linker molecule connecting tyrosine-phosphorylated proteins to enzymes which might lack their own SH2 domains. It has been shown that NCK forms a physical complex with the platelet-derived growth factor (PDGF) receptor and the epidermal growth factor receptor in a ligand-dependent manner (17, 21). To determine if NCK can mediate growth factor-stimulated early cellular responses, we tested the ability of NCK to activate transcription of the fos gene, a well-characterized...

Even though the retinoblastoma gene is one of the best-studied tumor suppressor genes, little is known about its functional role. Like all tumor suppressor gene products, the retinoblastoma protein (pRB) is thought to inhibit some aspect of cell proliferation. It also appears to be a cellular target of several DNA tumor virus-transforming proteins, such as adenovirus ElA, human papillomavirus E7, or simian virus 40 large T antigen. To help in the analysis of pRB, we have prepared a new set of anti-human pRB monoclonal antibodies. In addition to being useful reagents for the study of human pRB, these antibodies display several unexpected properties. They can be used to distinguish different subsets of the pRBs on the basis of their phosphorylation states. Some are able to recognize pRB homologs in other species, including mice, chickens, and members of the genus Xenopus. In addition, some of these antibodies can bind directly to other cellular proteins that, like pRB, were originally identified through their association with adenovirus ElA. These immunologically cross-reactive proteins include the p107 and p300 proteins, and their recognition by antibodies raised against pRB suggests that several members of the ElA-targeted cellular proteins form a structurally and functionally related family.Studies of the retinoblastoma gene (RBI) have greatly enforced the notion that tumor development is advanced not only by the gain of dominantly acting mutations but also by the loss of proteins that normally restrict cell growth (1,27,37). Inactivation of both alleles of the RBI gene has been found in all retinoblastoma tumors that have been studied to date, as well as in many small-cell lung carcinomas, osteosarcomas, breast carcinomas, soft tissue sarcomas, bladder carcinomas, and prostate carcinomas (3, 5, 7, 11, 13, 18-20, 26, 31-36, 41, 43). Evidence to support the notion that retinoblastoma protein (pRB) acts as a tumor suppressor comes from studies that show that the reintroduction of a wild-type pRB cDNA into cells without a functional retinoblastoma gene suppresses or delays tumorigenesis (4, 22).The RBI gene product (pRB) also forms a complex with several DNA tumor viral oncoproteins, including the adenovirus ElA proteins, the polyomavirus large T antigens, and human papillomavirus E7 proteins (6, 9, 30, 38). More important, genetic studies have shown that the sequences required for these oncoproteins to form complexes with pRB are also needed for oncogenesis (6,10,28,29,39,40). These studies strongly suggest that binding to pRB is essential for the oncogenic properties of these viral proteins. Moreover, the sequences essential for pRB to interact with ElA or simian virus 40 (SV40) T antigen are frequently mutated in the naturally occurring pRB mutants (21,23,24