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Src family kinases directly regulate JIP1 module dynamics and activation - PubMed

Src family kinases directly regulate JIP1 module dynamics and activation

Deepak Nihalani et al. Mol Cell Biol. 2007 Apr.

Abstract

JIP1 is a mammalian scaffold protein that assembles and participates in regulating the dynamics and activation of components of the mixed-lineage kinase-dependent JNK module. Mechanisms governing JIP1-JNK module regulation remain unclear. JIP1 is a multiply phosphorylated protein; for this reason, it was hypothesized that signaling by unidentified protein kinases or phosphatases might determine module function. We find that Src family kinases directly bind and tyrosine phosphorylate JIP1 under basal conditions in several naturally occurring systems and, by doing so, appear to provide a regulated signal that increases the affinity of JIP1 for DLK and maintains the JIP-JNK module in a catalytically inactive state.

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Figures

FIG. 1.
FIG. 1.

JIP1 interacts with SFKs in vivo. (A) Endogenous JIP1 was coimmunoprecipitated with endogenous Lyn, Fyn, or Yes from normal mouse brain lysates. Note that endogenous JIP1 that coimmunoprecipitated with the various SFKs was tyrosine phosphorylated. (B) Flag-JIP1 and the indicated SFKs were coexpressed in COS-7 cells. As in panel A, JIP1 coimmunoprecipitated with the indicated SFK and was tyrosine phosphorylated under these conditions. (C) JIP1 binds directly to Fyn in a tyrosine phosphorylation-dependent fashion. Immobilized His-Fyn was mixed with GST, GST-JIP1 or GST-JIP1 expressed in TKB1 cells. The GST-JIP1 pull-down complex was resolved by SDS-PAGE and immunoblotted using JIP1 antibody. Input of proteins was detected by Coomassie blue staining or immunoblotting. (D) Recombinant His-Fyn, GST-Fyn-SH2, GST-Fyn-SH3, or GST alone was run on SDS-PAGE, transferred to membranes, and overlaid with the indicated GST fusion proteins; bound JIP1 proteins were detected by immunoblotting. (E) Fyn directly phosphorylates JIP1. Purified recombinant His-JIP1 was mixed with either active His-Fyn or active GST-JNK (as a positive control), incubated in a kinase buffer containing [γ-32P]ATP at 30°C for 30 min, and then resolved by SDS-PAGE and autoradiography. The same blot was immunoblotted with JIP1 antibody to ensure that the phosphorylated protein is JIP1. Similar results were obtained in three independent experiments. (F) JIP1 is tyrosine phosphorylated in vivo. Flag-JIP1 was coexpressed with Fyn in COS-7 cells. Where indicated, at 24 h posttransfection cells were pretreated with PP2 (5 μM) or with sodium orthovanadate (50 μM) or both. JIP1 was immunoprecipitated from cell lysates, and immunoprecipitates were analyzed for JIP1 tyrosine phosphorylation by immunoblotting.

FIG. 2.
FIG. 2.

Binding of DLK to JIP1 but not oligomerization of DLK is increased in the presence of Fyn. (A) COS-7 cells were cotransfected with plasmids encoding Flag-JIP1, HA-DLK, and increasing amounts of Fyn. Cell lysates were immunoprecipitated with anti-JIP1 antibody, separated by SDS-PAGE, and immunoblotted with HA or Fyn antibody. Corresponding lysates were immunoblotted with Fyn, DLK, and JIP1 antibodies to evaluate the expression of these proteins. (B) Plasmids encoding Flag-DLK and HA-DLK were cotransfected with Myc-JIP1 and/or Fyn. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA antibody. These experiments were repeated three times with similar results. (C) Fyn catalytic activity is required for increased affinity of JIP1 for DLK. COS-7 cells were cotransfected with the indicated plasmids. Where noted, cells were treated with PP2 prior to cell lysis. JIP1 was immunoprecipitated from cell lysate, and immune complexes were separated by SDS-PAGE and immunoblotted. (D) Catalytic activity of Fyn is not required for association of Fyn with JIP1. COS-7 cells were cotransfected with plasmids encoding HA-DLK and Flag-JIP1 and with the indicated quantities of either wild-type Fyn or Fyn-KD (kinase-inactive mutant). JIP1 was immunoprecipitated from cell lysates, and immune complexes were separated by SDS-PAGE and immunoblotted with Fyn or HA antibodies.

FIG. 3.
FIG. 3.

SFKs directly phosphorylate JIP1 on tyrosine residues. (A) JIP1 peptides containing theoretical tyrosine phosphorylation sites (5) were used to generate a spot peptide array. As controls, mutant peptides were created in which tyrosine residues were replaced with phenylalanine. Recombinant His-JIP served as an additional control. The array was subjected to an in vitro kinase assay in the presence of recombinant Fyn or Yes. Asterisks indicate the predicted tyrosine residues within JIP1 peptides. (B to E) The indicated plasmids were expressed in COS-7 cells, and lysates were analyzed.

FIG. 4.
FIG. 4.

JIP tyrosine mutants attenuate the effect of Fyn on module activation. Myc-JNK was coexpressed with the indicated plasmids in COS-7 cells. JNK kinase catalytic activity was analyzed in kinase buffer containing the substrate GST-c-Jun. The relative activity from each sample was estimated quantitatively. Corresponding lysates were also analyzed for the expression of the indicated proteins. Results are means plus standard errors of the means for five independent experiments.

FIG. 5.
FIG. 5.

JIP1 is tyrosine phosphorylated in 3T3-L1 preadipocytes in an SFK-dependent fashion. (A) Survey of JIP1 expression and JIP1 tyrosine phosphorylation in neuronal cell lines (HN33 and NIEp115) or 3T3-L1 cells cultured in either DMEM containing calf serum (CS) for 2 days or in DMEM containing fetal bovine serum (FBS) for 8 days. (B to D) 3T3-L1 cells were grown for 2 days postconfluence in DMEM containing 10% CS and then stimulated with insulin (100 nM) for the indicated times. JIP1 was immunoprecipitated, and immune complexes were separated on SDS-PAGE and immunoblotted with the indicated antibodies. Immunoblot analysis of the corresponding cell lysates was performed to evaluate the presence of indicated proteins using their specific antibodies. In panel C, JIP1 immune complex was also subjected to JNK catalytic assay using GST-c-Jun as the substrate. These experiments were repeated five times with similar results. (D) Tyrosine phosphorylation of JIP1, total JNK activation, and IRS1 phosphorylation were analyzed after pretreatment of 3T3-L1 cells with the SFK inhibitors PP2 (5 μM) and SU6656 (1 μM).

FIG. 6.
FIG. 6.

Stimulation of neuronal cells with either reelin or kainic acid decreases Fyn-dependent JIP1 tyrosine phosphorylation. (A) HN33 neuronal cells were cotransfected with plasmids encoding Flag-JIP1 (1 μg), ApoER2 (0.5 μg), and Fyn (0.2 μg). At 24 h posttransfection, cells were treated with reelin for the indicated times. JIP1 was immunoprecipitated from the cell lysate, and the immune complexes were analyzed for JIP1 tyrosine phosphorylation and Fyn binding. (B) HN33 cells were cotransfected with Flag-JIP1 (1 μg) and Fyn (0.2 μg). At 24 h posttransfection, cells were treated with 100 μM kainic acid for the indicated times. Tyrosine phosphorylation of JIP1 was analyzed by immunoprecipitating JIP1 from cell lysates and immunoblotting with antiphosphotyrosine antibody. JNK catalytic activity was assayed by immunoprecipitating total JNK from lysate and incubating the immune complex with kinase buffer containing [γ-32P]ATP and GST-c-Jun.

FIG. 7.
FIG. 7.

Fyn attenuates JNK module activity by tyrosine phosphorylating JIP1. (A) Extracts were prepared from N2a cells treated with 100 μM kainic acid for 30 min or left untreated. JIP1 complexes were immunoprecipitated using JIP1 antibody and analyzed by immunoblotting with anti-DLK and antiphosphotyrosine antibodies. Immunoprecipitation with preimmune serum (PI) was used as control. (B and C) Mutation of tyrosine 431 in JIP1 induces JNK activation in N2a cells. Flag-JIP1(Y431F) (1 μg) or Flag-JIP1(R160G/P161G/Y431F) was expressed in N2a cells. At 24 h posttransfection, cells were treated with kainic acid for 30 min. Endogenous JNK catalytic activity was assayed in kinase buffer containing [γ-32P]ATP using GST-c-Jun as the substrate. The relative activity from each sample was quantified using a phosphorimager. This experiment was conducted three times with similar results. Results are means plus standard errors of the means.

FIG. 8.
FIG. 8.

Endogenous JIP1 module dynamics are altered after genetic deletion of Fyn in cell culture and in vivo. (A) SYF cells deficient in Src, Fyn, and Yes were transfected with plasmids expressing Fyn or a catalytically inactive Fyn (Fyn-KD) or with a vector control. JIP1 was immunoprecipitated from these cell lysates and examined. (B) JIP1 tyrosine phosphorylation and association of JIP1 with DLK is reduced in the brains of Fyn-deficient mice. JIP1 was immunoprecipitated from brain lysate of Fyn−/− or wild-type littermates using JIP1 antibody or preimmune control serum (PI). Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with DLK or antiphosphotyrosine antibodies. Endogenous JNK kinase catalytic activity was assayed as before. This experiment used two mice from each group and was repeated three times with similar results.

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