Full-length cDNA was generated with the SuperScript? Preamplification System (Invitrogen)

Full-length cDNA was generated with the SuperScript? Preamplification System (Invitrogen). sequence) and MCSP (using antibody against the extracellular portion of the MCSP core protein) as a model for ligands (Eisenmann et al., 1999). We have also shown that metastatic cells adherent on CS1 spread when the MCSP core protein was also engaged (Iida et al., 1995; Eisenmann et al., 1999). Wells coated with a recombinant GST fusion protein containing the CS1 41 integrin-binding domain of FN (GST-rIIIcs) promoted high levels of adhesion of both WM1552C and WM1341D cells (Fig. 3 B). Wells coated with the anti-MCSP mAb 9.2.27 also supported high adhesion levels of both WM1341D cells and WM1552C/MCSP transfectants (but not parental or mock WM1552C cells; Fig. 3 B). MCSP-expressing cells did not significantly spread on surfaces coated only with integrin-binding (GST-rIIIcs/IgG2a) or PG-binding (GST/mAb 9.2.27) substrates; however, chimeric GST-rIIIcs/mAb 9.2.27 (integrin/PG-binding) surfaces promoted extensive cell spreading (Fig. 3, C and D). MCSP expression enhances phosphorylation of FAK and ERK1/2 Because FAK is a key member of integrin-mediated signaling pathways and initial cell spreading is regulated partly through FAK activity in many cells (Guan, 1997; Schlaepfer et al., 1999), we tested whether MCSP could induce FAK activation. WM1341D cells were serum starved overnight and were then plated on the various surfaces as indicated (Fig. 4). Engaging integrin alone on surfaces coated with GST-rIIIcs/IgG2a caused a modest increase in the level of FAK LTV-1 pY397 that peaked at 30 min CDKN2AIP after plating (Fig. 4 A). By contrast, plating the WM1341D cells onto integrin/PG-binding substrata resulted in enhanced levels of FAK pY397 much greater than those observed in cells adherent only via 41 integrin. The kinetics of phosphorylation at FAK Y397 were similar on both substrates (Fig. 4 A). Plating cells on surfaces coated with GST/mAb 9.2.27, used to stimulate MCSP alone, did not result in increased FAK pY397 (Fig. 4 B), indicating that MCSP does not directly stimulate FAK phosphorylation. Open in a separate window Figure 4. MCSP stimulates FAK Y 397 and ERK1/2 phosphorylation in melanoma cells. Cells were serum starved overnight and LTV-1 plated on various chimeric substrata using the same concentrations described in Fig. 3 B. (A) WM1341D cells were allowed to adhere to the specified substrata at 37C for the indicated times, lysed with SDS sample buffer, and immunoblotted with total and phosphospecific FAK and ERK1/2 antibodies as indicated. (B) WM1341D cells were allowed to adhere to plates coated with mAb 9.2.27 and GST for the indicated times at 37C, lysed in SDS sample buffer, and lysates were evaluated by immunoblotting as in A. (C) WM1552C parental and transfectant cells were plated on the indicated substrata and allowed to adhere for 1 h at 37C. Cells were lysed in SDS sample LTV-1 buffer and analyzed for levels of FAK and ERK1/2 phosphorylation as in A. The ERK/MAPK pathway has been implicated in cell spreading and is one of the downstream effectors of activated FAK (Guan, 1997; Schlaepfer et al., 1999). We reprobed the blots to determine if there is a relationship between FAK pY397 and ERK1/2 phosphorylation in these cells. Phosphorylated ERK (pERK) 1/2 was easily detected in the VGP WM1341D cells (Fig. 4 A); it did not appreciably vary in these cells during the time course of the assay (0C60 min). The level of pERK1/2 was equal in cells plated on either the integrin- or the integrin/PG-binding substrates (Fig. 4 A), in contrast to what was observed with FAK phosphorylation. Engaging MCSP alone also had no effect on the level of pERK1/2 in these cells (Fig. 4 B). ERK1/2 is constitutively activated in suspended WM1341D cells (zero time point) and was not further phosphorylated after cell adhesion (Fig. 4, A and B). Therefore, FAK activation induced by adhesion of WM1341D cells does not appear to have an effect on the level of ERK phosphorylation. As was observed for the WM1341D cells, WM1552C/MCSP transfectants exhibited low levels.