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Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules.

Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, Rapoport TA, Margolis B - J. Cell Biol. (2001)

Bottom Line: Three proteins were found, the c-jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) JIP-1, JIP-2, and JIP-3, which are scaffolding proteins for the JNK signaling pathway.Coprecipitation experiments suggest that kinesin carries the JIP scaffolds preloaded with cytoplasmic (dual leucine zipper-bearing kinase) and transmembrane signaling molecules (the Reelin receptor, ApoER2).These results demonstrate a direct interaction between conventional kinesin and a cargo, indicate that motor proteins are linked to their membranous cargo via scaffolding proteins, and support a role for motor proteins in spatial regulation of signal transduction pathways.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. kverhey@hms.harvard.edu

ABSTRACT
The cargo that the molecular motor kinesin moves along microtubules has been elusive. We searched for binding partners of the COOH terminus of kinesin light chain, which contains tetratricopeptide repeat (TPR) motifs. Three proteins were found, the c-jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) JIP-1, JIP-2, and JIP-3, which are scaffolding proteins for the JNK signaling pathway. Concentration of JIPs in nerve terminals requires kinesin, as evident from the analysis of JIP COOH-terminal mutants and dominant negative kinesin constructs. Coprecipitation experiments suggest that kinesin carries the JIP scaffolds preloaded with cytoplasmic (dual leucine zipper-bearing kinase) and transmembrane signaling molecules (the Reelin receptor, ApoER2). These results demonstrate a direct interaction between conventional kinesin and a cargo, indicate that motor proteins are linked to their membranous cargo via scaffolding proteins, and support a role for motor proteins in spatial regulation of signal transduction pathways.

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Expression of kinesin dominant negative constructs causes mislocalization of endogenous JIP-1 protein. CAD cells were transiently transfected with plasmids encoding the HA-tagged KLC TPRs (A), HA-tagged PP5 TPRs (B), HA-tagged KLC truncation KLC-176 (C), or Myc-tagged KHC truncation KHC-891 (D). After differentiation, the expressed proteins were detected by indirect immunofluorescence microscopy using mAbs to the epitope tags (left). Endogenous JIP-1 protein was detected with an affinity-purified polyclonal antibody (right). Note that the background fluorescence has been enhanced to show the entire neuronal cell. Arrowheads denote tips of transfected cells; arrows denote tips of untransfected cells.
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Figure 5: Expression of kinesin dominant negative constructs causes mislocalization of endogenous JIP-1 protein. CAD cells were transiently transfected with plasmids encoding the HA-tagged KLC TPRs (A), HA-tagged PP5 TPRs (B), HA-tagged KLC truncation KLC-176 (C), or Myc-tagged KHC truncation KHC-891 (D). After differentiation, the expressed proteins were detected by indirect immunofluorescence microscopy using mAbs to the epitope tags (left). Endogenous JIP-1 protein was detected with an affinity-purified polyclonal antibody (right). Note that the background fluorescence has been enhanced to show the entire neuronal cell. Arrowheads denote tips of transfected cells; arrows denote tips of untransfected cells.

Mentions: In the first approach, we asked whether the COOH-terminal residues of JIP-1 that are required for binding to KLC are also important for localization of JIP-1 to the tips of neuronal processes. Myc-tagged full-length JIP-1, the NH2-terminal truncation JIP-1 (307–711), and the three mutant constructs JIP-1 (307–700), JIP-1 (P704A), and JIP-1 (Y709A) were localized in differentiated N1E 115 cells by indirect immunofluorescence using antibodies to the Myc tag. Untransfected control cells showed nonspecific background staining of the cell body (Fig. 4 A). Correct localization of Myc–JIP-1 fusion proteins was scored as specific staining of the neurite tips compared with the background staining of the cell body. As seen in Fig. 4B and Fig. C, full-length JIP-1 and the NH2-terminal truncation JIP-1 (307–711) localize to the tips of the neurites (arrowheads indicate tip staining) identical to the staining seen for endogenous JIP-1 (Fig. 5; Meyer et al. 1999; Yasuda et al. 1999; Kelkar et al. 2000; Pellet et al. 2000). Likewise, mutation of the proline residue, JIP-1 (P704A), had no effect on JIP-1 localization (Fig. 4 E). In contrast, JIP-1 with a deletion of the 11 COOH-terminal residues, JIP-1 (307–700), or mutation of the tyrosine residue, JIP-1 (Y709A), exhibited localization throughout the cell with no concentration at the neurite tips (Fig. 4D and Fig. F, arrows indicate staining of the cell body). These data demonstrate that the residues of JIP-1 that are critical for binding to the TPR motifs of KLC are also required for the proper subcellular localization of JIP-1 and support the model that JIP proteins are a cargo for kinesin.


Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules.

Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, Rapoport TA, Margolis B - J. Cell Biol. (2001)

Expression of kinesin dominant negative constructs causes mislocalization of endogenous JIP-1 protein. CAD cells were transiently transfected with plasmids encoding the HA-tagged KLC TPRs (A), HA-tagged PP5 TPRs (B), HA-tagged KLC truncation KLC-176 (C), or Myc-tagged KHC truncation KHC-891 (D). After differentiation, the expressed proteins were detected by indirect immunofluorescence microscopy using mAbs to the epitope tags (left). Endogenous JIP-1 protein was detected with an affinity-purified polyclonal antibody (right). Note that the background fluorescence has been enhanced to show the entire neuronal cell. Arrowheads denote tips of transfected cells; arrows denote tips of untransfected cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198804&req=5

Figure 5: Expression of kinesin dominant negative constructs causes mislocalization of endogenous JIP-1 protein. CAD cells were transiently transfected with plasmids encoding the HA-tagged KLC TPRs (A), HA-tagged PP5 TPRs (B), HA-tagged KLC truncation KLC-176 (C), or Myc-tagged KHC truncation KHC-891 (D). After differentiation, the expressed proteins were detected by indirect immunofluorescence microscopy using mAbs to the epitope tags (left). Endogenous JIP-1 protein was detected with an affinity-purified polyclonal antibody (right). Note that the background fluorescence has been enhanced to show the entire neuronal cell. Arrowheads denote tips of transfected cells; arrows denote tips of untransfected cells.
Mentions: In the first approach, we asked whether the COOH-terminal residues of JIP-1 that are required for binding to KLC are also important for localization of JIP-1 to the tips of neuronal processes. Myc-tagged full-length JIP-1, the NH2-terminal truncation JIP-1 (307–711), and the three mutant constructs JIP-1 (307–700), JIP-1 (P704A), and JIP-1 (Y709A) were localized in differentiated N1E 115 cells by indirect immunofluorescence using antibodies to the Myc tag. Untransfected control cells showed nonspecific background staining of the cell body (Fig. 4 A). Correct localization of Myc–JIP-1 fusion proteins was scored as specific staining of the neurite tips compared with the background staining of the cell body. As seen in Fig. 4B and Fig. C, full-length JIP-1 and the NH2-terminal truncation JIP-1 (307–711) localize to the tips of the neurites (arrowheads indicate tip staining) identical to the staining seen for endogenous JIP-1 (Fig. 5; Meyer et al. 1999; Yasuda et al. 1999; Kelkar et al. 2000; Pellet et al. 2000). Likewise, mutation of the proline residue, JIP-1 (P704A), had no effect on JIP-1 localization (Fig. 4 E). In contrast, JIP-1 with a deletion of the 11 COOH-terminal residues, JIP-1 (307–700), or mutation of the tyrosine residue, JIP-1 (Y709A), exhibited localization throughout the cell with no concentration at the neurite tips (Fig. 4D and Fig. F, arrows indicate staining of the cell body). These data demonstrate that the residues of JIP-1 that are critical for binding to the TPR motifs of KLC are also required for the proper subcellular localization of JIP-1 and support the model that JIP proteins are a cargo for kinesin.

Bottom Line: Three proteins were found, the c-jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) JIP-1, JIP-2, and JIP-3, which are scaffolding proteins for the JNK signaling pathway.Coprecipitation experiments suggest that kinesin carries the JIP scaffolds preloaded with cytoplasmic (dual leucine zipper-bearing kinase) and transmembrane signaling molecules (the Reelin receptor, ApoER2).These results demonstrate a direct interaction between conventional kinesin and a cargo, indicate that motor proteins are linked to their membranous cargo via scaffolding proteins, and support a role for motor proteins in spatial regulation of signal transduction pathways.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. kverhey@hms.harvard.edu

ABSTRACT
The cargo that the molecular motor kinesin moves along microtubules has been elusive. We searched for binding partners of the COOH terminus of kinesin light chain, which contains tetratricopeptide repeat (TPR) motifs. Three proteins were found, the c-jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) JIP-1, JIP-2, and JIP-3, which are scaffolding proteins for the JNK signaling pathway. Concentration of JIPs in nerve terminals requires kinesin, as evident from the analysis of JIP COOH-terminal mutants and dominant negative kinesin constructs. Coprecipitation experiments suggest that kinesin carries the JIP scaffolds preloaded with cytoplasmic (dual leucine zipper-bearing kinase) and transmembrane signaling molecules (the Reelin receptor, ApoER2). These results demonstrate a direct interaction between conventional kinesin and a cargo, indicate that motor proteins are linked to their membranous cargo via scaffolding proteins, and support a role for motor proteins in spatial regulation of signal transduction pathways.

Show MeSH
Related in: MedlinePlus