Universität zu Köln

Araki, Tsuyoshi, Na, Jianbo, Clemen, Christoph S., Williams, Jeffrey G. and Eichinger, Ludwig ORCID: 0000-0003-1594-6117 (2014). Identification of the Protein Kinases Pyk3 and Phg2 as Regulators of the STATc-Mediated Response to Hyperosmolarity. PLoS One, 9 (2). SAN FRANCISCO: PUBLIC LIBRARY SCIENCE. ISSN 1932-6203

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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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Araki, Tsuyoshi UNSPECIFIED UNSPECIFIED UNSPECIFIED Na, Jianbo UNSPECIFIED UNSPECIFIED UNSPECIFIED Clemen, Christoph S. UNSPECIFIED UNSPECIFIED UNSPECIFIED Williams, Jeffrey G. UNSPECIFIED UNSPECIFIED UNSPECIFIED Eichinger, Ludwig UNSPECIFIED orcid.org/0000-0003-1594-6117 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-445709
DOI:	10.1371/journal.pone.0090025
Journal or Publication Title:	PLoS One
Volume:	9
Number:	2
Date:	2014
Publisher:	PUBLIC LIBRARY SCIENCE
Place of Publication:	SAN FRANCISCO
ISSN:	1932-6203
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language DICTYOSTELIUM-DISCOIDEUM; OSMOTIC SHOCK; CELL POLARITY; STRESS; PATHWAY; RAP1; AGGREGATION; CHEMOTAXIS; ADHESION; INSIGHTS Multiple languages Multidisciplinary Sciences Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/44570