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Two Dictyostelium tyrosine kinase-like kinases function in parallel, stress-induced STAT activation pathways.

Araki T, Vu LH, Sasaki N, Kawata T, Eichinger L, Williams JG - Mol. Biol. Cell (2014)

Bottom Line: The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action.The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain.The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3.

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Affiliation: College of Life Sciences, Welcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.

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Characterization of the pyk2−, pyk3−, and double- strains. (A) Hyperosmotic stress–induced activation of STATc in parental Ax2 and pyk2−, pyk3−, and double- strains. Parental Ax2 cells and  cells were developed in shaken suspension for 4 h and then treated with sorbitol at 200 mM for the indicated times. Tyrosine phosphorylation of STATc was assayed by Western transfer, using phospho-STATc antibody CP22. The same blot was reprobed with total-STATc antibody 7H3 (unpublished data). The combined data from several such experiments are shown as fold relative to sorbitol-induced Ax2 (at 15 min) ± SD. Student's t test: *p < 0.05; **p < 0.01. (B) Nuclear enrichment of STATc in parental Ax2 and pyk2−, pyk3−, and double- strains. Cells developed as in A were treated with sorbitol at 200 mM for 5 min. The nuclear accumulation of STATc was assayed immunohistochemically, using total STATc antibody. KK2 PB–treated cells were used as control, as it is the vehicle for sorbitol. Bar, 10 μm.
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Figure 2: Characterization of the pyk2−, pyk3−, and double- strains. (A) Hyperosmotic stress–induced activation of STATc in parental Ax2 and pyk2−, pyk3−, and double- strains. Parental Ax2 cells and cells were developed in shaken suspension for 4 h and then treated with sorbitol at 200 mM for the indicated times. Tyrosine phosphorylation of STATc was assayed by Western transfer, using phospho-STATc antibody CP22. The same blot was reprobed with total-STATc antibody 7H3 (unpublished data). The combined data from several such experiments are shown as fold relative to sorbitol-induced Ax2 (at 15 min) ± SD. Student's t test: *p < 0.05; **p < 0.01. (B) Nuclear enrichment of STATc in parental Ax2 and pyk2−, pyk3−, and double- strains. Cells developed as in A were treated with sorbitol at 200 mM for 5 min. The nuclear accumulation of STATc was assayed immunohistochemically, using total STATc antibody. KK2 PB–treated cells were used as control, as it is the vehicle for sorbitol. Bar, 10 μm.

Mentions: In marked contrast to the response to DIF-1, for which the pyk2− mutant is totally unresponsive (Araki et al., 2012), the STATc response of pyk2− cells to hyperosmotic stress is only minimally diminished; relative to the parental Ax2 strain, there is just a 1-min delay in STATc activation, and a very similar plateau level is achieved (Figure 2A). This suggests that, in the case of stress but not DIF-1, another kinase is almost fully competent to subsume the function of Pyk2 as an activator of STATc. The logical candidate for this role is Pyk3; it has the most closely related kinase domain in the kinome (51% identity in the kinase domain, Supplemental Figure S4A), it is one of the five TKLs that autophosphorylate on tyrosine when expressed in E. coli, and two entirely independently constructed and analyzed strains for pyk3 (pyk3− strains, Supplemental Figure S2) show a 20–50% reduction in their sorbitol-induced activation of STATc (Figure 2A; Vu et al., 2014). This suggests that Pyk2 is partially able to substitute the function of Pyk3. However, the most striking result is observed with the double mutant, pyk2−/pyk3−. This strain is completely unresponsive to hyperosmotic stress–induced activation of STATc (Figure 2A).


Two Dictyostelium tyrosine kinase-like kinases function in parallel, stress-induced STAT activation pathways.

Araki T, Vu LH, Sasaki N, Kawata T, Eichinger L, Williams JG - Mol. Biol. Cell (2014)

Characterization of the pyk2−, pyk3−, and double- strains. (A) Hyperosmotic stress–induced activation of STATc in parental Ax2 and pyk2−, pyk3−, and double- strains. Parental Ax2 cells and  cells were developed in shaken suspension for 4 h and then treated with sorbitol at 200 mM for the indicated times. Tyrosine phosphorylation of STATc was assayed by Western transfer, using phospho-STATc antibody CP22. The same blot was reprobed with total-STATc antibody 7H3 (unpublished data). The combined data from several such experiments are shown as fold relative to sorbitol-induced Ax2 (at 15 min) ± SD. Student's t test: *p < 0.05; **p < 0.01. (B) Nuclear enrichment of STATc in parental Ax2 and pyk2−, pyk3−, and double- strains. Cells developed as in A were treated with sorbitol at 200 mM for 5 min. The nuclear accumulation of STATc was assayed immunohistochemically, using total STATc antibody. KK2 PB–treated cells were used as control, as it is the vehicle for sorbitol. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 2: Characterization of the pyk2−, pyk3−, and double- strains. (A) Hyperosmotic stress–induced activation of STATc in parental Ax2 and pyk2−, pyk3−, and double- strains. Parental Ax2 cells and cells were developed in shaken suspension for 4 h and then treated with sorbitol at 200 mM for the indicated times. Tyrosine phosphorylation of STATc was assayed by Western transfer, using phospho-STATc antibody CP22. The same blot was reprobed with total-STATc antibody 7H3 (unpublished data). The combined data from several such experiments are shown as fold relative to sorbitol-induced Ax2 (at 15 min) ± SD. Student's t test: *p < 0.05; **p < 0.01. (B) Nuclear enrichment of STATc in parental Ax2 and pyk2−, pyk3−, and double- strains. Cells developed as in A were treated with sorbitol at 200 mM for 5 min. The nuclear accumulation of STATc was assayed immunohistochemically, using total STATc antibody. KK2 PB–treated cells were used as control, as it is the vehicle for sorbitol. Bar, 10 μm.
Mentions: In marked contrast to the response to DIF-1, for which the pyk2− mutant is totally unresponsive (Araki et al., 2012), the STATc response of pyk2− cells to hyperosmotic stress is only minimally diminished; relative to the parental Ax2 strain, there is just a 1-min delay in STATc activation, and a very similar plateau level is achieved (Figure 2A). This suggests that, in the case of stress but not DIF-1, another kinase is almost fully competent to subsume the function of Pyk2 as an activator of STATc. The logical candidate for this role is Pyk3; it has the most closely related kinase domain in the kinome (51% identity in the kinase domain, Supplemental Figure S4A), it is one of the five TKLs that autophosphorylate on tyrosine when expressed in E. coli, and two entirely independently constructed and analyzed strains for pyk3 (pyk3− strains, Supplemental Figure S2) show a 20–50% reduction in their sorbitol-induced activation of STATc (Figure 2A; Vu et al., 2014). This suggests that Pyk2 is partially able to substitute the function of Pyk3. However, the most striking result is observed with the double mutant, pyk2−/pyk3−. This strain is completely unresponsive to hyperosmotic stress–induced activation of STATc (Figure 2A).

Bottom Line: The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action.The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain.The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Welcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.

Show MeSH
Related in: MedlinePlus