| Big mitogen activated protein kinase (BMK1/ERK5) is important for cell growth and survival. The goal of my research was to investigate mechanisms that regulate BMK1 signal transduction by identifying proteins that modify BMK1 signaling in response to EGF and fluid shear stress.; 1. I demonstrated that the adaptor protein 14-3-3beta bound to BMK1 directly through serine 486 within the C-terminus of BMK1, and inhibited BMK1 kinase activities stimulated by EGF. Mutation of serine 486 prevented the interaction with 14-3-3beta and enhanced BMK1 activity upon EGF stimulation. These data demonstrate an inhibitory function for 14-3-3beta binding to BMK1, and show that serine 486 phosphorylation represents a novel regulatory mechanism for BMK1.; 2. Because 14-3-3 is known to regulate MAPK activity I studied the effect of a dominant negative 14-3-3zeta on MAPK pathways in vascular smooth muscle cells (VSMC). I found that DN-14-3-3zeta inhibited Raf activation, but had no significant effect on activation of ERK1/2, JNK, p38MAPK or cell migration. The inability of DN-14-3-3zeta to regulate MAPK signaling in VSMC suggests the presence of redundant signaling pathways and/or failure to overcome endogenous 14-3-3.; 3. I demonstrated that the Grb-2 associated protein 1 (Gab1) positively regulated BMK1 activation in response to EGF, that was dependent on Gab1 recruitment to the EGF receptor and phosphorylation by c-Src. I showed that phosphatase SHP-2, a prominent Gab1 associated protein, was a negative mediator of BMK1 signaling. These findings identify Gab1 as a new mediator of BMK1 signaling, downstream of the EGF receptor and c-Src.; 4. To study how flow activates BMK1, I investigated the role of Gab1 in BMK1 signaling in ECs. I demonstrated that flow stimulated Gab1 tyrosine phosphorylation and Gab1 interaction with SHP-2. Furthermore, both Gab1 and SHP-2 acted as negative regulators of BMK1 signaling by flow.; These data provide novel information regarding mechanisms of BMK1 activation and define two different pathways for BMK1 activation by flow and the EGF receptor. |
