A novel 14-kilodalton protein interacts with the mitogen-activated proteinkinase scaffold MP1 on a late endosomal/lysosomal compartment

We have identified a novel, highly conserved protein of 14 kD copurifying w ith late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes /lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activa ted protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-reg ulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S -transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasm a membrane-targeted p14 causes mislocalization of coexpressed MP1. In addit ion, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro. The interaction between p14 and MP1 suggests a MAPK scaffolding activity lo calized to the cytoplasmic surface of late endosomes/lysosomes, thereby com bining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.