Identification of the protein kinases Pyk3 and Phg2 as regulators of the STATc-mediated response to hyperosmolarity.

Cellular adaptation to changes in environmental osmolarity is crucial for cell survival. In Dictyostelium, 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 Ca(2+)-dependent, of which many signaling components have yet to be identified. The duanyu1813c stress signalling pathway feeds back on itself by upregulating the expression of duanyu1813c and genes. Based on microarray studies we chose two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators of duanyu1813c phosphorylation and generated single and double knock-out mutants to them. Transcriptional regulation of duanyu1813c and duanyu1813c 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 duanyu1813c dependent genes in response to sorbitol, 8-Br-cGMP and the Ca(2+) liberator BHQ. Also, 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 The protein tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits 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 duanyu1813c by either directly or indirectly activating it. Our results add further complexities to the regulation of duanyu1813c, which presumably ensure its optimal activation in response to different environmental cues.

KEYWORDS: {{ getKeywords(articleDetailText.words) }}