Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase

Tsuyoshi Araki 2 Judith Langenick 2 Marianne Gamper 0 1 Richard A. Firtel 1 Jeffrey G. Williams 2 0 Biomedical Research Foundation (SBF) , Lauchefeld 31, CH-9548 Matzingen , Switzerland 1 University of California , San Diego, Natural Sciences Building Room 6111, 9500 Gilman Drive, La Jolla, CA 92093-0380 , USA 2 University of Dundee, College of Life Sciences , Dow Street, Dundee DD1 5EH , UK STATc becomes tyrosine phosphorylated and accumulates in the nucleus when Dictyostelium cells are exposed to the prestalk cell inducer Differentiation inducing factor 1 (DIF-1), or are subjected to hyper-osmotic stress. We show that the protein tyrosine phosphatase PTP3 interacts directly with STATc and that STATc is refractory to activation in PTP3 overexpressing cells. Conversely, overexpression of a dominant inhibitor of PTP3 leads to constitutive tyrosine phosphorylation and ectopic nuclear localisation of STATc. Treatment of cells with DIF-1 or exposure to hyper-osmotic stress induces a decrease in biochemically assayable PTP3 activity and both agents also induce serine-threonine phosphorylation of PTP3. These observations suggest a novel mode of STAT activation, whereby serine-threonine phosphorylation of a cognate protein tyrosine phosphatase results in the inhibition of its activity, shifting the phosphorylation-dephosphorylation equilibrium in favour of phosphorylation. INTRODUCTION STAT proteins are important regulators of metazoan gene expression (Bromberg and Darnell, 2000). They are activated when diverse signalling pathways are stimulated, but the various JAK-STAT pathways form the paradigm. In response to the binding of a cytokine to its receptor, an associated tyrosine kinase of the JAK family phosphorylates a STAT at a unique site near its C terminus. This leads, via reciprocal phosphotyrosine:SH2 domain interactions, to dimerisation of the STAT and the STAT dimers accumulate in the nucleus (Reich and Liu, 2006). Dictyostelium cells use STAT signalling to regulate several aspects of their differentiation (Williams, 2003). Extracellular cAMP signalling activates STATa, which can function as either a repressor or an activator of specific gene expression (Araki et al., 1998; Fukuzawa and Williams, 2000). DIF-1 is a chlorinated hexaphenone that induces differentiation of one of the prestalk cell subtypes, pstO cells (Thompson and Kay, 2000). At the slug stage, STATc is nuclear localised in pstO cells, where it acts to prevent ectopic expression of a marker of pstA cell differentiation (Fukuzawa et al., 2001). Addition of DIF-1 to cells early in development leads to the premature tyrosine phosphorylation, dimerisation and nuclear accumulation of STATc. Nuclear accumulation of STATc in response to DIF-1 is regulated at the level of nuclear export (Fukuzawa et al., 2003). In uninduced cells, the effect of a nuclear import signal, located near the N terminus of STATc, is negated by a DIF-1-regulated nuclear export signal located near the centre of the protein. The balance between import and export activity seems to be linked to the homodimerisation that is triggered by phosphorylation of STATc on tyrosine residue 922. The mechanism by which STATc becomes tyrosine phosphorylated is unknown. There are no apparent *These authors contributed equally to this work Author for correspondence (e-mail: ) Dictyostelium homologues of the class of tyrosine kinases that modify metazoan STATs (Goldberg et al., 2006). There are, however, an unusually large number of tyrosine kinase-like enzymes that perhaps subsume their function. In mammals, STAT1 and STAT3 are activated by specific cytokines but hyper-osmotic stress is also an activator (Gatsios et al., 1998). Similarly, STATc accumulates in the nucleus rapidly when cells are subjected to hyper-osmotic stress (Araki et al., 2003). Tyrosine phosphorylation and nuclear localisation of STATc are maintained for at least 30 minutes. By contrast, the fraction of STATc protein that is tyrosine phosphorylated and nuclear localised after DIF-1 treatment reaches a sharp peak at 3-5 minutes of treatment and STATc is then de-phosphorylated and exits the nucleus. One likely explanation for the temporal disparity, between the stress and the DIF-1 responses, is a difference in de-phosphorylation kinetics. In metazoa TC45, the nuclear isoform of the T-cell protein tyrosine phosphatase (TC-PTP), serves to de-phosphorylate STAT1 and this causes it to re-localise to the cytoplasm (ten Hoeve et al., 2002). TC-PTP-null cells are also defective in STAT3 dephosphorylation but TC45 may not be the only phosphatase involved; because the cytosolic form of PTP de-activates STAT3 when overexpressed (Tanuma et al., 2000). STAT5 activation is normal in TC-PTP-null cells and here there is evidence for direct interaction of STAT5 with the non-receptor tyrosine phosphatases SHP2 (Yu et al., 2000) and with PTP1B (Aoki and Matsuda, 2000). Thus, the metazoan STATs, which differ significantly in their mechanisms of nuclear accumulation (Reich and Liu, 2006), are also heterogeneous in their modes and sites of de-activation. The two processes are of course intimately inter-related; a nuclear protein must actively shuttle between nucleus and cytoplasm if a cytosolic tyrosine phosphatase is to serve to de-activate it. The tyrosine phosphatase that catalyses de-phosphorylation of STATc is unknown. Dictyostelium encodes three PTPs, all of which are predicted to be non-transmembrane proteins (Howard et al., 1992; Howard et al., 1994; Gamper et al., 1996). PTP1 and PTP2null strains show only minor defects in development, but PTP1 and PTP2 overexpressing strains develop aberrantly and contain an altered spectrum of tyrosine phosphorylated proteins. PTP1 is a negative regulator of STATa tyrosine phosphorylation but the failure to detect a direct interaction between the two proteins, using a substrate-trapping form of PTP1, suggests that PTP1 acts indirectly, at some point upstream of STATa (Early et al., 2001). The third phosphatase, PTP3, is divergent in several otherwise highly conserved amino acid residues and the bacterially produced enzyme has a very low intrinsic phosphatase activity (Gamper et al., 1996). Analysis of a tagged version of the protein suggests it to be partly cytosolic and partly nuclear (Gamper et al., 1999). In the Ax3derived strain JH10 there are two copies of the PTP3 gene. It was possible to disrupt one copy, but the second copy was refractory to disruption (Gamper et al., 1996). Antisense inhibition also proved ineffective. Although PTP3 seems to be essential for cell viability, overexpression studies have yielded some insights into its developmental functions. The PTP3 overexpression strain grows slowly and forms large aggregation streams; in addition, many structures arrest development at the mound stage and there are changes in the tyrosine phosphorylation level of several proteins (Gamper et al., 1996; Gamper et al., 1999). In parental cells, (...truncated)