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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.

View Article: PubMed Central - PubMed

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

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Pyk3 and STATc physically interact. (A) Analysis of the interaction of GST-Pyk2 and GST-Pyk3 with STATc. Ax2 cells were developed in suspension for 4 h and then left untreated or exposed to sorbitol as in Figure 2A. Cells were lysed, and the extracts were subjected to pull-down assay using GST-Pyk2-WT or GST-Pyk2-K880A, its kinase-dead form, and GST-Pyk3-WT or GST-Pyk3-K1084A. Bound STATc was detected using total STATc antibody 7H3. The blot was reprobed with a GST antibody as a loading control. (B) Tyrosine phosphorylation–dependent GST-Pyk3 binding to STATc. GST-Pyk3 was bound to glutathione beads, and the beads were either left untreated or digested with TC-PTP. A parallel reaction was performed on GST-Pyk3 beads in the presence of sodium orthovanadate, an inhibitor of the enzyme, left untreated or induced with sorbitol, and assayed as in A. The blot was reprobed with a GST antibody as a loading control, and the same samples were reanalyzed with a general phosphotyrosine antibody 4G10. (C) Pull-down analysis of Pyk3 by parental and mutant forms of STATc. Myc-Pyk2 OE or myc-Pyk3 OE cell lysate from control cells, or cells induced with sorbitol, were subjected to pull-down assay using GST-STATc or its R831A and Y922F mutant forms as in A. The blot was reprobed with a GST antibody as a loading control.
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Figure 6: Pyk3 and STATc physically interact. (A) Analysis of the interaction of GST-Pyk2 and GST-Pyk3 with STATc. Ax2 cells were developed in suspension for 4 h and then left untreated or exposed to sorbitol as in Figure 2A. Cells were lysed, and the extracts were subjected to pull-down assay using GST-Pyk2-WT or GST-Pyk2-K880A, its kinase-dead form, and GST-Pyk3-WT or GST-Pyk3-K1084A. Bound STATc was detected using total STATc antibody 7H3. The blot was reprobed with a GST antibody as a loading control. (B) Tyrosine phosphorylation–dependent GST-Pyk3 binding to STATc. GST-Pyk3 was bound to glutathione beads, and the beads were either left untreated or digested with TC-PTP. A parallel reaction was performed on GST-Pyk3 beads in the presence of sodium orthovanadate, an inhibitor of the enzyme, left untreated or induced with sorbitol, and assayed as in A. The blot was reprobed with a GST antibody as a loading control, and the same samples were reanalyzed with a general phosphotyrosine antibody 4G10. (C) Pull-down analysis of Pyk3 by parental and mutant forms of STATc. Myc-Pyk2 OE or myc-Pyk3 OE cell lysate from control cells, or cells induced with sorbitol, were subjected to pull-down assay using GST-STATc or its R831A and Y922F mutant forms as in A. The blot was reprobed with a GST antibody as a loading control.

Mentions: The interaction between Pyk2 and STATc can readily be demonstrated using Pyk2-GST fusion protein, produced in E. coli, as a binding substrate for STATc in a “pull-down” assay (Araki et al., 2012). The same holds true for Pyk3. In both cases there is a basal level of STATc binding to Pyk2 and Pyk3 that decreases somewhat after exposure to sorbitol (Figure 6A). Thus binding of STATc to the two kinases is semiconstitutive. The reason for a decrease is unclear, but one possibility is that some change in STATc, such as serine/threonine phosphorylation, is induced by high osmolarity, and this inhibits stress-induced binding to STATc in some unknown way. Interaction between Pyk2 and STATc occurs when STATc binds to a site or sites of tyrosine autophosphorylation on Pyk2 (Araki et al., 2012). When a similar experiment is performed using GST-Pyk3 and its kinase-dead form rather than GST-Pyk2, it yields identical results (Figure 6A).


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)

Pyk3 and STATc physically interact. (A) Analysis of the interaction of GST-Pyk2 and GST-Pyk3 with STATc. Ax2 cells were developed in suspension for 4 h and then left untreated or exposed to sorbitol as in Figure 2A. Cells were lysed, and the extracts were subjected to pull-down assay using GST-Pyk2-WT or GST-Pyk2-K880A, its kinase-dead form, and GST-Pyk3-WT or GST-Pyk3-K1084A. Bound STATc was detected using total STATc antibody 7H3. The blot was reprobed with a GST antibody as a loading control. (B) Tyrosine phosphorylation–dependent GST-Pyk3 binding to STATc. GST-Pyk3 was bound to glutathione beads, and the beads were either left untreated or digested with TC-PTP. A parallel reaction was performed on GST-Pyk3 beads in the presence of sodium orthovanadate, an inhibitor of the enzyme, left untreated or induced with sorbitol, and assayed as in A. The blot was reprobed with a GST antibody as a loading control, and the same samples were reanalyzed with a general phosphotyrosine antibody 4G10. (C) Pull-down analysis of Pyk3 by parental and mutant forms of STATc. Myc-Pyk2 OE or myc-Pyk3 OE cell lysate from control cells, or cells induced with sorbitol, were subjected to pull-down assay using GST-STATc or its R831A and Y922F mutant forms as in A. The blot was reprobed with a GST antibody as a loading control.
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Figure 6: Pyk3 and STATc physically interact. (A) Analysis of the interaction of GST-Pyk2 and GST-Pyk3 with STATc. Ax2 cells were developed in suspension for 4 h and then left untreated or exposed to sorbitol as in Figure 2A. Cells were lysed, and the extracts were subjected to pull-down assay using GST-Pyk2-WT or GST-Pyk2-K880A, its kinase-dead form, and GST-Pyk3-WT or GST-Pyk3-K1084A. Bound STATc was detected using total STATc antibody 7H3. The blot was reprobed with a GST antibody as a loading control. (B) Tyrosine phosphorylation–dependent GST-Pyk3 binding to STATc. GST-Pyk3 was bound to glutathione beads, and the beads were either left untreated or digested with TC-PTP. A parallel reaction was performed on GST-Pyk3 beads in the presence of sodium orthovanadate, an inhibitor of the enzyme, left untreated or induced with sorbitol, and assayed as in A. The blot was reprobed with a GST antibody as a loading control, and the same samples were reanalyzed with a general phosphotyrosine antibody 4G10. (C) Pull-down analysis of Pyk3 by parental and mutant forms of STATc. Myc-Pyk2 OE or myc-Pyk3 OE cell lysate from control cells, or cells induced with sorbitol, were subjected to pull-down assay using GST-STATc or its R831A and Y922F mutant forms as in A. The blot was reprobed with a GST antibody as a loading control.
Mentions: The interaction between Pyk2 and STATc can readily be demonstrated using Pyk2-GST fusion protein, produced in E. coli, as a binding substrate for STATc in a “pull-down” assay (Araki et al., 2012). The same holds true for Pyk3. In both cases there is a basal level of STATc binding to Pyk2 and Pyk3 that decreases somewhat after exposure to sorbitol (Figure 6A). Thus binding of STATc to the two kinases is semiconstitutive. The reason for a decrease is unclear, but one possibility is that some change in STATc, such as serine/threonine phosphorylation, is induced by high osmolarity, and this inhibits stress-induced binding to STATc in some unknown way. Interaction between Pyk2 and STATc occurs when STATc binds to a site or sites of tyrosine autophosphorylation on Pyk2 (Araki et al., 2012). When a similar experiment is performed using GST-Pyk3 and its kinase-dead form rather than GST-Pyk2, it yields identical results (Figure 6A).

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