Takanori Goi

An EGF receptor/Ral‐GTPase signaling cascade regulates c‐Src activity and substrate specificity

c‐Src is a membrane‐associated tyrosine kinase that can be activated by many types of extracellular signals, and can regulate the function of a variety of cellular protein substrates. We demonstrate that epidermal growth factor (EGF) and β‐adrenergic receptors activate c‐Src by different mechanisms leading to the phosphorylation of distinct sets of c‐Src substrates. In particular, we found that EGF receptors, but not β2‐adrenergic receptors, activated c‐Src by a Ral‐GTPase‐dependent mechanism. Also, c‐Src activated by EGF treatment or expression of constitutively activated Ral‐GTPase led to tyrosine phosphorylation of Stat3 and cortactin, but not Shc or subsequent Erk activation. In contrast, c‐Src activated by isoproterenol led to tyrosine phosphorylation of Shc and subsequent Erk activation, but not tyrosine phosphorylation of cortactin or Stat3. These results identify a role for Ral‐GTPases in the activation of c‐Src by EGF receptors and the coupling of EGF to transcription through Stat3 and the actin cytoskeleton through cortactin. They also show that c‐Src kinase activity can be used differently by individual extracellular stimuli, possibly contributing to their ability to generate unique cellular responses.

An Eps Homology (EH) Domain Protein That Binds to the Ral-GTPase Target, RalBP1

Ral proteins constitute a family of small GTPases that can be activated by Ras in cells. In the GTP-bound state, Ral proteins bind to RalBP1, a GTPase-activating protein for CDC42 and Rac GTPases. We have used the two-hybrid system in yeast to clone a cDNA for a novel ∼85-kDa protein that can bind to an additional site on RalBP1. This newly identified protein contains an Eps homology (EH) domain, which was first detected in the epidermal growth factor (EGF) receptor substrate Eps15. Recently, the EH domain of Eps15 has been shown to bind to proteins containing an asparagine-proline-phenylalanine motif. Moreover, EH domains have been found in proteins involved in endocytosis and/or actin cytoskeleton regulation. The RalBP1 associated Eps-homology domain protein, Reps1, is tyrosine-phosphorylated in response to EGF stimulation of cells. In addition, Reps1 has the capacity to form a complex with the SH3 domains of the adapter proteins Crk and Grb2, which may link Reps1 to an EGF-responsive tyrosine kinase. Thus, Reps1 may coordinate the cellular actions of activated EGF receptors and Ral-GTPases.

Ral-Specific Guanine Nucleotide Exchange Factor Activity Opposes Other Ras Effectors in PC12 Cells by Inhibiting Neurite Outgrowth

ABSTRACT Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only ∼20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.

Ral-GTPase influences the regulation of the readily releasable pool of synaptic vesicles

ABSTRACT The Ral proteins are members of the Ras superfamily of GTPases. Because they reside in synaptic vesicles, we used transgenic mice expressing a dominant inhibitory form of Ral to investigate the role of Ral in neurosecretion. Using a synaptosomal secretion assay, we found that while K+-evoked secretion of glutamate was normal, protein kinase C-mediated enhancement of glutamate secretion was suppressed in the mutant mice. Since protein kinase C effects on secretion have been shown to be due to enhancement of the size of the readily releasable pool of synaptic vesicles docked at the plasma membrane, we directly measured the refilling of this readily releasable pool of synaptic vesicles after Ca2+-triggered exocytosis. Refilling of the readily releasable pool was suppressed in synaptosomes from mice expressing dominant inhibitory Ral. Moreover, we found that protein kinase C and calcium-induced phosphorylation of proteins thought to influence synaptic vesicle function, such as MARCKS, synapsin, and SNAP-25, were all reduced in synaptosomes from these transgenic mice. Concomitant with these studies, we searched for new functions of Ral by detecting proteins that specifically bind to it in cells. Consistent with the phenotype of the transgenic mice described above, we found that active but not inactive RalA binds to the Sec6/8 (exocyst) complex, whose yeast counterpart is essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. These findings demonstrate a role for Ral-GTPase signaling in the modulation of the readily releasable pool of synaptic vesicles and suggest the possible involvement of Ral-Sec6/8 (exocyst) binding in modulation of synaptic strength.

Reduced expression of deleted colorectal carcinoma (DCC) protein in established colon cancers

Using a bacterial fusion protein, a deleted colorectal carcinoma (DCC)-specific monoclonal antibody (MAb) 127-22 was established. Although MAb 127-22 reacted with almost all normal tissues, it did not react or only weakly reacted with many cancer cell lines, including colonic cancer lines, in flow cytometry. In Western immunoblots, the MAb reacted with a single 190-kDa molecule in a myeloma line Ara-10 extract. This component was scarcely detected in colonic cancer cell lines. Immunoblots of samples from 25 pairs of colonic cancers and adjacent normal tissues and from five adenoma tissues revealed that all normal colonic and adenoma tissues significantly expressed the DCC protein, whereas colonic cancer tissues showed poor expression. These results indicate not only deletion of and lowered mRNA expression of the DCC gene, but also marked reduction of DCC protein occurred in colonic cancer tissues. In addition, colonic cancer patients with liver metastasis expressed significantly lower levels of DCC than those without, suggesting the prognostic value of DCC expression.

The Relationship Between Metastatic Potential and the Expression of Sialyl Lewis A (CA 19–9) and Sialyl Lewis X Antigens in Colorectal Cancer

The relationship between metastatic potential and the expression of sialyl Lea or sialyl Lex antigens was studied in 128 advanced colorectal cancer. Hematogenous metastasis was observed more often in the Grade II-III group of sialyl Lea or sialyl Lex staining than in Grade 0-1. The recurrence rates in the Grade II-III group of sialyl Lex staining was significantly higher than those in the Grade 0-1, and the prognosis in the Grade II-III group of sialyl Lex staining was significantly poorer than that in the Grade 0-1. These results suggested that sialyl Lea and sialyl Lex antigens in colorectal cancer might be involved in the hematogenous metastasis.

DNA Index and p53 Immunoreaction in Colorectal Cancer and Its Relationship to Prognosis

The DNA ploidy pattern and p53 immunoreactivity was studied in colorectal cancers and the results correlate with prognosis. There was no significant correlation between the DNA ploidy pattern and clinicopathological findings. However, patients with aneuploid tumor ran significantly poorer than diploid ones. The 5- and 10-year survival rates were 60.4% and 53.4% for those with diploid tumor. On the other hand, p53 immunoreactivity was found 57.1% of 203 colorectal cancers. The 5-year survival rate was 62.8% of patients with p53-positive tumors, and 74.2% for ones with p53-negative tumors: there was a significant differece between these two groups. We suggest therefore that DNA ploidy pattern and p53 immunoreactivity may possible be a useful prognostic marker of colorectal cancers.