05-372 Sigma-AldrichAnti-GRB2 Antibody, clone 3F2

We identified a novel protein kinase C (PKC)alpha-dependent signal to extracellular signal-regulated kinase (ERK)1/2 in mouse osteoclasts and Chinese hamster ovary (CHO) cells, specifically activated by the alphaVbeta3 integrin. It involves translocation (i.e. activation) of PKCalpha from the cytosol to the membrane and/or the Triton X-100-insoluble subcellular fractions, with recruitment into a complex with alphaVbeta3 integrin, growth factor receptor-bound protein (Grb2), focal adhesion kinase (FAK) in CHO cells and proline-rich tyrosine kinase (PYK2) in osteoclasts. Engagement of alphavbeta3 integrin triggered ERK1/2 phosphorylation, but the underlying molecular mechanism was surprisingly independent of the well known Shc/Ras/Raf-1 cascade, and of phosphorylated MAP/ERK kinase (MEK)1/2, so far the only recognized direct activator of ERK1/2. In contrast, PKCalpha was involved in ERK1/2 activation because inhibition of its activity prevented ERK1/2 phosphorylation. The tyrosine kinase c-Src also contributed to ERK1/2 activation, however, it did not interact with PKCalpha in the same molecular complex. The alphaVbeta3/PKCalpha complex formation was fully dependent upon the intracellular calcium concentration ([Ca2+]i), and the use of the intracellular Ca2+ chelator 1,2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraaceticacidtetra (acetoxymethyl) ester (BAPTA-AM) also inhibited PKCalpha translocation and ERK1/2 phosphorylation. Functional studies showed that alphaVbeta3 integrin-activated PKCalpha was involved in cell migration and osteoclast bone resorption, but had no effect on the ability of cells to attach to LM609, suggesting a role in events downstream of alphaVbeta3 integrin engagement.

Stimulation of the T cell antigen receptor (TCR) activates signaling pathways involving protein kinases, phospholipase Cgamma1, and Ras. How these second messengers interact to initiate distal activation events is an area of intense scrutiny. In this report, we confirm that TCR ligation results in phosphorylation of Sos, a guanine nucleotide exchange factor for Ras. This requires expression of both the CD45 tyrosine phosphatase and the Lck protein tyrosine kinase and depends upon signaling via protein kinase C. In contrast to previous studies examining requirements for Sos phosphorylation following insulin and epidermal growth factor receptor engagement, we show that TCR-induced phosphorylation of Sos does not require activation of the mitogen-activated protein kinase/extracellular-signal regulated kinase (MEK/ERK) pathway. However, the basal phosphorylation of Sos in T cells is affected by either MEK or MEK-dependent kinases. Although Sos phosphorylation results in its dissociation from Grb2 following insulin stimulation in Chinese hamster ovary cells, TCR engagement on the Jurkat T cell line fails to elicit a similar effect. These data demonstrate that the kinases responsible for Sos phosphorylation differ following ligation of various cell surface receptors and that the consequences of Sos phosphorylation relies, at least in part, on sites of its phosphorylation.

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.

The protein ASH (for abundant Src homology), composed of one Src homology region (SH) 2 and two SH3 domains, was cloned by screening human and rat cDNA libraries with an oligonucleotide probe directed to a consensus sequence of the SH2 domains. The rat-derived ASH peptide was comprised of 217 amino acids with a molecular mass of 25-28 kDa and was found to be ubiquitous in rat tissues. A human cDNA clone was also found to code for part of the same protein, suggesting that ASH is common to human and rat. The amino acid sequence of ASH was strikingly similar to Sem-5, the product of a nematode cell-signaling gene, and ASH is most probably a mammalian homologue of Sem-5. ASH bound in vitro to phosphotyrosine-containing proteins, including activated epidermal growth factor receptor, the ASH SH2 domain being responsible for the binding. Induced expression of an antisense ASH cDNA led to a reduction in cell growth. Considering these observations and the structural homology to Sem-5, ASH is likely to function as a ubiquitous signal transducer, possibly resembling Sem-5, which communicates between a receptor protein tyrosine kinase and a Ras protein.