A Novel 14-Kilodalton Protein Interacts with the Mitogen-Activated Protein Kinase Scaffold Mp1 on a Late Endosomal/Lysosomal Compartment

Abstract

We have identified a novel, highly conserved protein of 14 kD copurifying with 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-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal–regulated 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 plasma membrane–targeted p14 causes mislocalization of coexpressed MP1. In addition, 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 localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Receptor-specific requirements for anthrax toxin delivery into cells

“…This effect may be due to less efficient pore formation (Fig. 1b) or LF unfolding (25) under more neutral pH conditions; or instead might be due to a reduction in the accessibility of MEK1, which might be concentrated on scaffolds on late endosomes (34), under these conditions. Our results are consistent with different receptor-specific pH requirements for pore formation in the endosomal pathway.…”

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

“…These include the effectors Rain1 on the Golgi and the activated Raf-MEK-ERK cascade on endosomes [83,84]. Further examples include scaffolds for the Raf-MAPK cascade; whilst KSR is recruited to the cell surface, Sef is localised on the Golgi and p14-MP1 on endosomes [85][86][87][88].…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…Our recent observations have suggested a potential role for the MP1-p14 complex in coupling MAP kinase signaling to Rho [126]. MP1 is a small protein (14 kDa) identified as a MEK1 and ERK1 binding partner [127]; p14 is a protein tightly associated with late endodomes and lysosomes that was subsequently found to interact with high affinity with MP1 [128][129][130]. Specifically, we have found that MP1-p14 is important for PAK phosphorylation of MEK1 and ERK activation during adhesion to fibronectin, and that siRNA-mediated depletion of the complex causes a spreading defect in fibroblasts in concert with the formation of dense circumferential F-actin and large vinculin containing adhesions [126].…”

“…For instance, MP1 associates tightly with p14, a protein tightly associated with late endosomes and lysosomes [129,130], and this localization is important for ERK activation in response to EGF [128]. MP1-p14 appears important for focal adhesion remodeling during cell spreading on fibronectin [126].…”