Signaling pathways and structural domains required for phosphorylation of EMS1/cortactin

The structural characteristics of EMS1 (human cortactin) suggest that it ma y link signaling events to reorganization of the actin cytoskeleton, Intere stingly, the EMS1 gene is commonly amplified and overexpressed in several h uman cancers, which may alter their invasive or metastatic properties. An 8 0 to 85-kDa mobility shift of EMS1 correlates with an alteration in subcell ular distribution and is Likely to represent an important regulatory event. In HEK 293 cells, epidermal growth factor treatment or cell detachment ind uced this shift, and this was blocked by the mitogen-activated protein/extr acellular signal-regulated kinase kinase (MEK) inhibitor PDD98059, Furtherm ore, expression of a constitutively active form of MEK induced the shift, i ndicating that MEK activation was both sufficient and necessary for this mo dification. The epidermal growth factor-induced shift correlated with incre ased phosphorylation on serine and threonine residues of the same tryptic p hosphopeptides detected under basal conditions. Deletion of the helical-pro line-rich. region of the protein blocked the mobility shift and EMS1 phosph orylation. In vitro kinase assays demonstrated that the extracellular signa l-regulated kinases represent candidate kinases for this region, although o ther MEK-regulated enzymes must also participate, These data identify MEK a s an important intermediate involved in EMS1 phosphorylation and highlight the helical-proline-rich region as a key regulatory domain.