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Article Figures & Data

Figures

  • Fig. 1.

    Tec is involved in the signaling pathway of GM-CSF receptor. (A) UT-7 cells (1 × 107) were cultured in the starvation medium for 12 hours and then stimulated with 10 ng/mL of human GM-CSF (+) for 5 minutes or left unstimulated (−). Tec was immunoprecipitated from each fraction (αTec), subjected to 7.5% SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody (αP-Tyr). Total cell lysates (TCL; 10 μg/lane) and the immunoprecipitates by normal rabbit serum (NRS) prepared from the same set of cells were also analyzed. The position of Tec is indicated by an arrow. The molecular weight standards (×10−3) are shown at the left. The same membrane was reblotted with anti-Tec serum to show the amounts of Tec precipitated (lower panel). (B) UT-7 cells were stimulated with GM-CSF (10 ng/mL) for 0, 1, 5, 10, or 20 minutes as indicated at the top. Tec was immunoprecipitated from each fraction (1 × 107 cells), and was immunoblotted with anti-phosphotyrosine antibody (αP-Tyr) or anti-Tec serum (αTec). (C) Tec was immunoprecipitated from 1 × 107 of UT-7 cells with (+) or without (−) 5 minutes of GM-CSF stimulation, and was subjected to an in vitro kinase assay. Autophosphorylation of pp70Tec is shown. (D) Specific kinase activity of the Tec protein (32P-incorporation/protein amount) with (+) or without (−) the GM-CSF stimulation was calculated by densitometric analysis and shown as arbitrary units. (E) Tec was immunoprecipitated from UT-7 cells (1 × 107), with (GM) or without (−) the GM-CSF stimulation (10 ng/mL), metabolically labeled with [32P]orthophosphate (37 MBq/mL), and was analyzed by 7.5% SDS-PAGE. The proteins were blotted onto a PVDF membrane, and heated in 1 N KOH to decrease the backgrounds of serine- and threonine-phosphorylation. The position of Tec is indicated. (F) pp70Tec in (E) was subjected to the phosphoamino acid analysis. The positions of free phosphate (Pi), phosphoserine (p-Ser), phosphothreonine (p-Thr), and phosphotyrosine (p-Tyr) are indicated at the right.

  • Fig. 2.

    Tec is involved in the cytokine-driven activation of c-fos proto-oncogene. (A) BA/F3-hGMRαβ cells (1 × 107) were transfected with the pfos/luc reporter plasmid (2 μg) together with 5 μg each of the pSRα (Vector), pSRα-Syk (Syk), pSRα-Lyn (Lyn), pSRα-Jak2 (Jak), or pSRα-Tec (Tec). After 5 hours of incubation in cytokine-free medium, the cells were further cultured for 5 hours without (no factor) or with 5 ng/mL of human GM-CSF (+GM-CSF) or 25 U/mL of mouse IL-3 (+IL-3). Luciferase activity was assayed in each fraction and calculated as relative light units (RLU)/min/μg of protein. The mean value plus SD of the luciferase activities in triplicate samples from each fraction is shown as arbitrary units. (B) BA/F3-hGMRαβ cells were transfected with pSRα-Syk, pSRα-Lyn, pSRα-Jak2, or pSRα-Tec, and cultured for 24 hours in the presence of IL-3. Total cell lysates (10 μg/lane) were prepared from each set (+) and untransfected BA/F3-hGMRαβ cells (−), and were immunoblotted with the antibodies against the corresponding kinases.

  • Fig. 3.

    Tec can phosphorylate Jak2 in both mammalian and insect cells. (A) Jak2KE was immunoprecipitated from 2 × 106 of 293 cells expressing Jak2KE with (T) or without (−) Tec. Total cell lysates (TCL, 10 μg/lane) and anti-Jak2 immunoprecipitates (Jak IP) were electrophoresed and probed with anti-phosphotyrosine antibody (αP-Tyr) or anti-Jak2 serum (αJak2). The positions of Jak2 (Jak2), Tec (Tec), and the Ig heavy chain (IgH) are indicated at the right. (B) Jak2KE was immunoprecipitated from Sf21 cells infected with Jak2KEexpressing baculovirus (JE) alone or in combination with Tec-expressing (T) or TecKM-expressing (TM) virus. The immunoprecipitates were separated through 7.5% SDS-PAGE and probed with anti-phosphotyrosine antibody (αP-Tyr) or anti-Jak2 serum (αJak2). The positions of Jak2 (Jak2) and Tec (Tec) are indicated at the right. (C) Jak2 was immunoprecipitated from Sf21 cells expressing Jak2 (J) or Jak2KE (JE) either alone or in combination with Tec (T). The immunoprecipitates were incubated with [γ-32P]ATP and the synthetic Jak2-substrate, and subjected to Tricine-SDS-PAGE. Phosphorylation of the Jak2-substrate is shown. (D) Total cell lysates (TCL: 10 μg/lane) and the anti-Jak2 immunoprecipitates (Jak IP) were prepared from parental BA/F3 cells (P) and two BA/F3 clones (1 and 2) stably expressing Tec▵SH3, and immunoblotted with αP-Tyr Ab (upper panel) or anti-Jak2 serum (lower panel). The position of Jak2 is indicated at the right. The positions of molecular weight standards (×10−3) are also shown at the left.

  • Fig. 4.

    Jak2 can phosphorylate Tec at Tyr-518. (A) The kinase-dead TecKM (TM) was expressed in 293 cells either alone or in combination with Jak2 (Jak2) or Lyn (Lyn) kinase. Tec was immunoprecipitated from each fraction, and probed with anti-phosphotyrosine antibody (αP-Tyr) or anti-Tec serum (αTec). (B) TecKM (TM), TecKM▵SH3 (TM▵3), or TecKM,YF (TM,518F) was expressed in 293 cells either alone (−) or in combination with Jak2 (J) or Lyn (L). Tec was immunoprecipitated from each fraction, and blotted with anti-phosphotyrosine antibody (αP-Tyr) or anti-Tec serum (αTec). The positions of full-length Tec (T) and SH3-deleted (▵SH3) forms are indicated at the right. (C) The amino acid sequences of the Tec-family kinases, surrounding the tyrosine residues corresponding to Tyr-518 in mouse Tec, are compared. The asterisk indicates the position of the phosphorylated tyrosine. At the left shown are the numbers of amino acid positions of mouse Tec,2 human Btk,10 11 mouse Emt/Itk/Tsk, 9,41,42 human Bmx,43 and human Txk.44 (D) pSRα (V), pSRα-Tec (T), or pSRα-TecKM (TM) was transfected into 293 cells with or without pSRα-Jak2 (J). Tec was immunoprecipitated from each fraction, and incubated with [γ−32P]ATP without exogenous substrates. Autophosphorylation of pp70Tec in each sample is shown.

  • Fig. 5.

    Tec can constitutively associate with Jak2 in Sf21 cells. Sf21 cells were infected with baculovirus expressing Tec (T), Jak2 (J), TecKM (TM), and Jak2KE(JE) in the combinations indicated at the top. Jak2 was immunoprecipitated from each cells lysed by the 0.1%-lysis buffer (αJak IP), and probed with either anti-Tec serum (αTec) or anti-Jak2 serum (αJak2). Total cell lysates (TCL: 10 μg/lane) of each fraction were also probed with anti-Tec serum to estimate the expression level of Tec.

  • Fig. 6.

    Cytokine-driven pathways to the c-fosproto-oncogene. When activated by Lyn, Tec phosphorylates “Substrate X” and triggers the signaling pathway linked to the c-fosactivation. Tec and Jak2 can trans-phosphorylate each other. The biological significance of this phosphorylation in the context of c-fos activation mechanism is not settled yet. Jak2 is required for the appropriate function of “Substrate X” as well as for the phosphorylation of STATs. The STATs activation may also have some roles in the regulation of the c-fos transcription.