RIN1 regulates cell migration through RAB5 GTPases and ABL tyrosine kinases

Abstract

Stimulation of a receptor tyrosine kinase (RTK), such as EGFR, leads to RAS activation followed by RIN1 activation. RIN1, in turn, activates RAB5 family GTPases, as well as ABL tyrosine kinases. As expected, RIN1 expression directly correlates with RAB5-mediated EGFR endocytosis. We previously showed that normal receptor endocytosis and internalized EGFR fate also depend on the ability of RIN1 to concomitantly activate ABL tyrosine kinases, consistent with the established role of ABL kinases in cytoskeleton remodeling and the growing evidence that such remodeling plays a role in endocytic processes. Here we report that growth factor-directed cell migration, a physiological process that involves receptor endocytosis and actin remodeling, also requires the ability of RIN1 to coordinate RAB5 GTPase and ABL tyrosine kinase pathways.

Keywords: ABL; BIN1; EGFR; RAB5; RIN1; endocytosis.

Figure 1. RIN1^QM shows enhanced RAB5 Activation. (A, top) Immunoblots of activated RAB5 in RIN1 and RIN1^QM HeLa cells untreated (−) or stimulated with 100 ng/ml EGF for 5 min (+). Alternate lanes were left blank to prevent merging of the lanes. Note that EGF addition samples are not alternating. (Bottom) Whole cell lysates of RIN1 and RIN1^QM HeLa cells from above, immunoblotted for total RAB5, Tubulin and RIN1. (B) Quantification of relative activated RAB5, normalized to total RAB5 from (A). The RIN1^QM protein includes multiple carboxy terminal tags that increase it molecular mass.

Figure 2. RIN1→RAB5 signaling axis promotes directional migration, while the RIN1→ABL axis blocks cell migration. (A) Migration across a transwell over one day toward control medium (white bar) or 100 ng/ml EGF medium (black bar) for vector, RIN1 and RIN1^E574A HeLa cells. (B) Migration across a transwell, as in (A), for vector, RIN1 and RIN1^QM HeLa cells. (C) Immunoblot showing RIN1 protein levels in vector, RIN1, RIN1^E574A and RIN1^QM HeLa cells.