Intersectin Can Regulate the Ras/MAP Kinase Pathway Independent of Its Role in Endocytosis

Abstract

We previously identified intersectin, a multiple EH and SH3 domain-containing protein, as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. More recently, we identified mammalian Sos, a guanine-nucleotide exchange factor for Ras, as an intersectin SH3 domainbinding partner. We now demonstrate that overexpression of intersectin's SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4°C or with phenylarsine oxide, treatments previously established to inhibit EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation. Src homology 3 (SH3)1 domains are 50-to 70-amino acid protein modules that mediate protein-protein interactions through binding to specific proline-rich peptide sequences. The recruitment to the plasma membrane of the Ras guanine-nucleotide exchange factor, mammalian son-of-sevenless (mSos), via the actions of the adaptor protein Grb2, is a well characterized example of a functional role for SH3 domain-mediated interactions (1-6). Through its SH3 domains, Grb2 interacts with proline-rich sequences in mSos, and through its SH2 domain, Grb2 interacts with SHC or activated growth factor receptors, thereby recruiting mSos to the membrane where it can activate Ras (7,8). Activated Ras initiates a phosphorylation cascade culminating in phosphorylation and activation of the p42/p44 MAP kinases (Erk-1 and Erk-2).SH3 domain-mediated protein-protein interactions also function broadly in vesicular trafficking, particularly in clathrin-mediated endocytosis (9). For example, several laboratories have recently identified and cloned a novel protein, variously referred to as intersectin (frog and man) (10, 11), Dap160 (fruit fly) (12), Ese (mouse) (13), and EHSH (rat) (14), which contains two Eps15 homology (EH) domains and four or five tandem SH3 domains. Through its SH3 domains, intersectin interacts with the endocytic enzyme dynamin, and through its EH domains, intersectin binds the epsins (10), which are recently identified EH domain-interacting proteins that localize to clathrin-coated pits (15). Intersectin is also localized to clathrin-coated pits in neurons and non-neuronal cells (16), and it has been proposed that the protein functions as a molecular scaffold in clat...

Identification and characterization of EBP, a novel EEN binding protein that inhibits Ras signaling and is recruited into the nucleus by the MLL-EEN fusion protein

“…Intersectin has been shown to regulate the Ras/mitogen-activated protein (MAP) kinase pathway and activate Elk-1 transcription factor, resulting in oncogenic transformation of rodent cells. [38][39][40] Overexpression of a dominant negative mutant with SH3 domains of intersectin repressed the activation of Ras and Elk-1 39 and failed to induce transformation. 40 In contrast to intersectin, the expression For personal use only.…”

“…Because EBP displays high structural similarity with the multiple SH3 domains in the C-terminal of intersectin, which has been reported to interact with Sos and activate Elk-1 activation, 39,40 we also investigated the effect of EBP on the downstream signaling of Ras by performing luciferase assay using Gal4-Elk-1 transreporter. Expression of RasQ61L, a constitutively active mutant, enhanced the activation of Elk-1 transreporter by 6-fold (P Ͻ .001, ANOVA) ( Figure 7C).…”

Dual treatment with COX-2 inhibitor and sodium arsenite leads to induction of surface Fas Ligand expression and Fas-Ligand-mediated apoptosis in human melanoma cells

“…In contrast, COX-2 inhibitors have been shown to suppress transcription of several matrix metalloproteinases and to upregulate Dynamin-2 gene expression, which controls protein export and endocytosis in the cell [72,82,83]. General inhibition of endocytosis in melanomas by phenylarsine oxide (PAO; 1 μM) [84], which appears to suppress recycling membrane FasL, also substantially increased surface expression of FasL (Fig. 8D).…”

Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed

“…11 The C-terminus granzyme B cleavage product of ITSN, comprising five consecutive SH3 domains, acts as dominant negative on Ras/Erk1/2 prosurvival signaling. 49 Moreover, the opposing effects of p38 activation on Erk1/2 50 may further contribute to the high p38/ Erk1/2 MAPK activity ratio, and subsequently to abnormal EC proliferation. These observations strongly suggest that down-regulation of ITSN and the presence of the NH 2 -terminal EH ITSN fragment with EC proliferative potential are key for formation of obliterative and plexiform lesions in severe PAH and that the EH ITSN , a biologically active protein fragment, present in the lungs of experimental models of PAH and human PAH specimens, may be the trigger for the formation of pulmonary lesions.…”