SH3P2 in complex with Cbl and Src.

In this report, we describe SH3P2, an SH3-domain containing protein, as a novel Cbl-interacting molecule that is a substrate of tyrosine kinase Src. We identified a specific polyproline motif of Cbl responsible for binding of SH3P2 and Src, and observed mutual sequestration of Src and SH3P2 from monomer Cbl molecules. In adherent cells, SH3P2 associated with Cbl and fibrilar actin and was localized at focal contacts in fibroblasts as well as at the apical part of podosome rings in differentiated osteoclasts. Our data implicate that SH3P2, a novel component of adhesion sites, is involved in Cbl and Src-mediated pathways.

Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172).

Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

LERK-2, a binding protein for the receptor-tyrosine kinase ELK, is evolutionarily conserved and expressed in a developmentally regulated pattern.

We have isolated and characterized cDNA clones that encode the rat homologue of a binding protein, LERK-2, for the receptor tyrosine kinase, elk. The cDNAs contain an open reading frame of 1527 nucleotides capable of encoding a protein 345 amino acid residues in length. The nucleotide sequence of the present clones is > 90% identical to the previously identified human LERK-2 cDNA, and the predicted proteins encoded by the rat and human clones are identical at 95% of amino acid residues. Recombinant proteins expressed from the rat cDNAs bind to elk with high affinity, similar to recombinant human LERK-2 and an endogenously-expressed rat elk-binding protein. Expression of the rat LERK-2 mRNA was detected in embryonic brain, kidney, lung, skeletal muscle, thymus, liver, and heart, and diminished in the early post-natal period. Significant LERK-2 mRNA expression in the young adult rat was restricted to the lung, kidney, heart and testes.

A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner.

p47v-crk (v-Crk), a transforming gene product containing Src homology (SH)-2 and -3 domains, induces an elevated level of tyrosine phosphorylation of several cellular proteins. Among these proteins, a 125-135 kDa protein (p130) shows marked phosphorylation at tyrosines and tight association with v-Crk, suggesting a direct signal mediator of v-Crk. Here we report the molecular cloning of rat p130 by immunoaffinity purification. The p130 is a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding motifs of v-Crk. Immunochemical analyses revealed that p130 is highly phosphorylated at tyrosines during transformation by p60v-src (v-Src), as well as by v-Crk, forming stable complexes with these oncoproteins. The p130 behaves as an extremely potent substrate of kinase activity included in the complexes and it is a major v-Src-associated substrate of the Src kinase by partial peptidase mapping. Subcellular fractionation demonstrated that the cytoplasmic p130 could move to the membrane upon tyrosine phosphorylation. The p130 (designated Cas for Crk-associated substrate) is a common cellular target of phosphorylation signal via v-Crk and v-Src oncoproteins, and its unique structure indicates the possible role of p130Cas in assembling signals from multiple SH2-containing molecules.

Functional divergence of the MAP kinase pathway. ERK1 and ERK2 activate specific transcription factors.

Growth factor-receptor interactions at the cell surface eventually leading to the transcriptional activation of immediate early genes is mediated by the mitogen-activated protein kinase (MAP kinase/MAPK) cascade. Here we show that overexpression of extracellular signal-regulated kinase 1 (ERK1) cDNA, encoding p44mapk, results in the activation of Elk-1, the serum response factor accessory protein. We also show that overexpression of ERK2, encoding p42mapk, activates Myc, but not Elk-1. Therefore, the MAP kinase cascade diverges with at least one specific target for each MAP kinase isoform and provides a novel mechanism for differential regulation of this signaling pathway.

Characterization of the gene encoding a folate-binding protein expressed in human placenta. Identification of promoter activity in a G-rich SP1 site linked with the tandemly repeated GGAAG motif for the ets encoded GA-binding protein.

The gene encoding a folate-binding protein (FBP) expressed in human placenta has been cloned by screening a genomic library with the KB cell FBP complementary DNA. This gene, contained in a 10-kilobase EcoRI fragment of this genomic clone, has 5 exons, 4 introns, the AATAA polyadenylation signal in the 3'-untranslated region, and a 5'-flanking sequence which contains the promoter elements, all of which span approximately 5 kilobases. Transcription initiation was mapped by RNase protection to a site 73 base pairs downstream from a G-rich sequence linked to a tandemly repeated GGAAG sequence which is a motif that the ets oncogene encoded GA-binding protein (GABP) transcription factor binds. Gel-shift and supershift mobility assays indicate that the G-rich sequence and the ets motif bind specifically to SP1 and GABP, respectively. These cis regulatory elements in tandem drive expression of the chloramphenicol acetyltransferase reporter gene in transiently transfected mouse 3T3 cells. The location of these elements upstream of transcription initiation in this gene, which lacks an appropriately located TATA box promoter, indicates that this SP1-GA binding region most probably regulates expression of this placental FBP. The gene encoding this placental FBP has been assigned the FBP/PL-1 gene because it is a member of a multigene family that includes a gene encoding a FBP expressed in both KB cells and placenta and its unprocessed pseudogene.