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A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

Wunderlich W, Fialka I, Teis D, Alpi A, Pfeifer A, Parton RG, Lottspeich F, Huber LA - J. Cell Biol. (2001)

Bottom Line: We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients.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.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, A-1030 Vienna, Austria.

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.

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Recruitment of MEK1 to the p14–MP1 complex. Extracts from HeLa cells transiently transfected with HA-MEK1 together with X-p14 or MP1-myc6 or both were subjected to immunoprecipitation using polyclonal anti-myc antibodies. Input and immunoprecipitates were used for immunoblot analysis using anti-HA, anti-myc, and anti-Xpress antibodies.
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Figure 11: Recruitment of MEK1 to the p14–MP1 complex. Extracts from HeLa cells transiently transfected with HA-MEK1 together with X-p14 or MP1-myc6 or both were subjected to immunoprecipitation using polyclonal anti-myc antibodies. Input and immunoprecipitates were used for immunoblot analysis using anti-HA, anti-myc, and anti-Xpress antibodies.

Mentions: Surprisingly, our coimmunoprecipitation experiments from cell extracts had contrary results. Although we could coimmunoprecipitate HA-tagged MEK1 together with p14 and MP1 (Fig. 11), we did not succeed in recruiting Flag-tagged ERK1 to the p14–MP1–MEK1 complex (data not shown).


A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

Wunderlich W, Fialka I, Teis D, Alpi A, Pfeifer A, Parton RG, Lottspeich F, Huber LA - J. Cell Biol. (2001)

Recruitment of MEK1 to the p14–MP1 complex. Extracts from HeLa cells transiently transfected with HA-MEK1 together with X-p14 or MP1-myc6 or both were subjected to immunoprecipitation using polyclonal anti-myc antibodies. Input and immunoprecipitates were used for immunoblot analysis using anti-HA, anti-myc, and anti-Xpress antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195784&req=5

Figure 11: Recruitment of MEK1 to the p14–MP1 complex. Extracts from HeLa cells transiently transfected with HA-MEK1 together with X-p14 or MP1-myc6 or both were subjected to immunoprecipitation using polyclonal anti-myc antibodies. Input and immunoprecipitates were used for immunoblot analysis using anti-HA, anti-myc, and anti-Xpress antibodies.
Mentions: Surprisingly, our coimmunoprecipitation experiments from cell extracts had contrary results. Although we could coimmunoprecipitate HA-tagged MEK1 together with p14 and MP1 (Fig. 11), we did not succeed in recruiting Flag-tagged ERK1 to the p14–MP1–MEK1 complex (data not shown).

Bottom Line: We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients.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.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, A-1030 Vienna, Austria.

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.

Show MeSH
Related in: MedlinePlus