A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.
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.
Research Institute of Molecular Pathology, A-1030 Vienna, Austria.
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W
Wunderlich
I
I
Fialka
D
D
Teis
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A
Alpi
A
A
Pfeifer
R G
RG
Parton
F
F
Lottspeich
L A
LA
Huber
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002451:Cell Compartmentation; D017124:Conserved Sequence; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C426009:LAMTOR2 protein, mouse; C114559:Lamtor3 protein, mouse; D008565:Membrane Proteins; D011506:Proteins