Open Access Research article

The interaction of Kinesin-1 with its adaptor protein JIP1 can be regulated via proteins binding to the JIP1-PTB domain

Tomoko Satake12, Karin Otsuki1, Yumi Banba1, Jun Suenaga1, Hisashi Hirano3, Yuko Yamanaka3, Shigeo Ohno1 and Syu-ichi Hirai14*

Author Affiliations

1 Department of Molecular Biology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan

2 Molecular Medicine and Informatics Doctoral Program, Yokohama City University Graduate School of Medicine,Yokohama 236-0004, Japan

3 Department of Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, Yokohama 230-0045, Japan

4 Department of Biology, Wakayama Medical University School of Medicine, Wakayama 641-0011, Japan

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BMC Cell Biology 2013, 14:12  doi:10.1186/1471-2121-14-12

Published: 4 March 2013

Additional files

Additional file 1: Figure S1:

JNK activity does not affect JIP1 binding to kinesin-1. (A). Co-precipitation of GFP-JIP1 mutants and endogenous JNK in Neuro2a cells. The same samples as shown in Fig. 2A were analyzed by WB with the indicated antibodies. (B). Neuro2a cells transiently expressing TAP-JIP1 were treated with SP600125 (20 μM) or DMSO (vehicle) for 2 hours. Lysates were precipitated with SA. SA, proteins bound to streptavidin sepharose. (C). RFP-JIP1-Neuro2a cells were transfected with expression vectors for T7-MEKK1 or pUC8 (empty vector). Lysates were immunoprecipitated with anti-RFP antibody and analyzed by WB with the indicated antibodies. *: non-specific band.

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Additional file 2: Figure S2:

Sequence alignments of the JIP1 PTB domain. (A) JIP1 PTB domain aligned with Shc- and Dab1-PTB domains. All sequences are from mouse. The gray shading indicates the amino acids corresponding to JIP1 V581, F642 and F687. (B). Alignment of JIP1 PTB domains between species. The gray shading indicates the amino acids corresponding mouse JIP1 V581, F642 and F687.

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Additional file 3: Figure S3:

Mouse JIP3 peptides identified by Mascot search (Matrix Science). Amino acid sequence of mouse JIP3. Capital letters shaded gray indicate the peptides identified by Mascot search.

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Additional file 4: Figure S4:

The effect of JIP3 knockdown was offset by JIP3-WT but not JIP3-LZ4A. (A). Lysates prepared from RFP-JIP1-Neuro2a cells expressing shRNA targeting JIP3 (shJIP3), non-silencing control (NS) shRNA, TAP-JIP3-WT, or TAP-JIP3-LZ4A, in the combination as indicated were immunoprecipitated with anti-RFP antibody and analyzed by WB with the indicated antibodies. Input, cell lysate used for the immunoprecipitation assay. IP:RFP, immunoprecipitated proteins. (B). Quantification of kinesin-1 binding by RFP-JIP1 in (A). Kinesin-1 binding was normalized to the amount of precipitated RFP-JIP1 and input of kinesin-1. Results of two independent experiments are shown. (C). Differentiated RFP-JIP1-Neuro2a cells were transfected with shRNA vectors (NS or shJIP3) containing a GFP expression cassette. Arrowheads indicate the neurite tips of transfected cells. Scale bar = 20 μm. (D). Quantification of the relative fluorescence of RFP-JIP1 in the neurite tip. *: p < 0.03. Error bars indicate ± SEM. n = 50 for each construct.

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