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© The Rockefeller University Press, 0021-9525/2001//765 $5.00
The Journal of Cell Biology, Volume 152, Number 4, , 2001 765-776

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

^a Research Institute of Molecular Pathology, A-1030 Vienna, Austria
^b Institute for Molecular Bioscience, Centre for Microscopy and Microanalysis, and Department of Physiology and Pharmacology, University of Queensland, Queensland 4072, Brisbane, Australia
^c Max-Planck-Institute of Biochemistry, D-82152 Martinsried, Germany
Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.43-179-87-15343-179-730-885

huber{at}nt.imp.univie.ac.at

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.

Key Words: signal transduction scaffold • MEK • ERK • subcellular localization • endocytosis

© 2001 The Rockefeller University Press

W. Wunderlich and I. Fialka contributed equally to this work.

Abbreviations used in this paper: 2DGE, two-dimensional gel electrophoresis; EE, early endosome; EGFR, EGF receptor; ERK, extracellular signal–regulated kinase; GST, glutathione S-transferase; LBPA, lysobisphosphatidic acid; LE, late endosome; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAPK/ERK kinase; MP1, MEK partner 1; PAR, protease-activated receptor; pI, isoelectric point; PKA, protein kinase A; PNS, postnuclear supernatant.

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