Identification of the Protein Kinases Pyk3 and Phg2 as Regulators of the STATc-Mediated Response to Hyperosmolarity

Abstract

Cellular adaptation to changes in environmental osmolarity is crucial for cell survival. In Dictyostelium, STATc is a key regulator of the transcriptional response to hyperosmotic stress. Its phosphorylation and consequent activation is controlled by two signaling branches, one cGMP- and the other Ca2+-dependent, of which many signaling components have yet to be identified. The STATc stress signalling pathway feeds back on itself by upregulating the expression of STATc and STATc-regulated genes. Based on microarray studies we chose two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators of STATc phosphorylation and generated single and double knock-out mutants to them. Transcriptional regulation of STATc and STATc dependent genes was disturbed in pyk3−, phg2−, and pyk3−/phg2− cells. The absence of Pyk3 and/or Phg2 resulted in diminished or completely abolished increased transcription of STATc dependent genes in response to sorbitol, 8-Br-cGMP and the Ca2+ liberator BHQ. Also, phospho-STATc levels were significantly reduced in pyk3− and phg2− cells and even further decreased in pyk3−/phg2− cells. The reduced phosphorylation was mirrored by a significant delay in nuclear translocation of GFP-STATc. The protein tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits STATc, is inhibited by stress-induced phosphorylation on S448 and S747. Use of phosphoserine specific antibodies showed that Phg2 but not Pyk3 is involved in the phosphorylation of PTP3 on S747. In pull-down assays Phg2 and PTP3 interact directly, suggesting that Phg2 phosphorylates PTP3 on S747 in vivo. Phosphorylation of S448 was unchanged in phg2− cells. We show that Phg2 and an, as yet unknown, S448 protein kinase are responsible for PTP3 phosphorylation and hence its inhibition, and that Pyk3 is involved in the regulation of STATc by either directly or indirectly activating it. Our results add further complexities to the regulation of STATc, which presumably ensure its optimal activation in response to different environmental cues.

Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

“…As recently demonstrated, in Dictyostelium cells the Ser448 and Ser747 phosphorylation of the protein-tyrosine phosphatase-3 (PTP3) is induced by a variety of stresses resulting in inhibition of its phosphatase activity and, consequently, maintenance of its substrate, STATc, activity [40]. In agreement with the data from literature, the results showed that after hyperosmotic stress both expression and activity (phosphorylation status at Tyr694) of STAT5 were increased (Fig.…”

“…It is well described that when mammalian cells are subjected to osmotic or oxidative stress they respond to such stresses through activation of JAK/STAT signaling pathways and maintenance of its activity through inhibition of protein tyrosine phosphatases such as PTP3. As recently demonstrated, in Dictyostelium cells the Ser448 and Ser747 phosphorylation of the protein-tyrosine phosphatase-3 (PTP3) is induced by a variety of stresses resulting in inhibition of its phosphatase activity and, consequently, maintenance of its substrate, STATc, activity [ 40 ]. In agreement with the data from literature, the results showed that after hyperosmotic stress both expression and activity (phosphorylation status at Tyr694) of STAT5 were increased ( Fig.…”

TwoDictyosteliumtyrosine kinase–like kinases function in parallel, stress-induced STAT activation pathways

“…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 null 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 ).…”

“…Thus drugs that elevate intracellular calcium levels activate STATc, and at least one of them, 2,5,-di(tert-butyl)-1-4-benzohydroquinone (BHQ), leads to increased serine phosphorylation at one of the regulatory sites: S747 ( Figure 1 ). Analysis of a gene disruptant suggests that Phg2, a serine/threonine-specific member of the TKL group, lies in the pathway that represses PTP3 activity by modifying it on S747 ( Figure 1 ; Vu et al. , 2014 ).…”