Fig. 4.

TM4SF10 functionally alters cell shape and Fyn activity in MDCK cells. A: MDCK cells stably expressing GFP, TM4SF10-GFP, or Fyn-GFP, and coexpression of TM4SF10-GFP in Fyn-GFP expressing cells were examined by phase-contrast microscopy to monitor changes in colony formation behavior. B: MDCK cells expressing the same GFP fusion proteins as shown in A were plated at low density to visual morphology of individual cells. Representative cell images are shown and percentage of cells with extensions are graphed below (*P < 0.001 compared with all other samples, **P < 0.001 compared with Fyn-GFP). C: MDCK cells stably expressing Fyn-GFP were cotransfected with TM4SF10-GFP or ADAP-GFP, followed by immunoblotting with an anti-phospho-tyrosine antibody (P-Tyr) to show total cellular tyrosine kinase activity. D: schematic diagram of Fyn kinase and two major phosphorylation sites: the activating phospho-tyrosine⁴²¹ (pY⁴²¹) of the kinase domain and the inactivating phospho-tyrosine⁵³² (pY⁵³²) that induces conformation changes that block the SH2 domain. E: effect of TM4SF10-GFP on Fyn phosphorylation using Western blotting for total Fyn and phospho-specific antibodies. The additional band on the Src pY⁴²¹ blot (which is not detected by the Fyn-specific antibody) is likely another Src family kinase since these antibodies will detect the same phospho-epitope on other Src kinases; however, the degree of phosphorylation of this other band does not appear to change with TM4SF10-GFP expression.