Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling

Abstract

SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are classified mainly as serine-threonine kinases. STATs are transcription factors that dimerize via phosphotyrosine-SH2 domain interactions. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We show that in a null mutant for Pyk2, a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc. Conversely, overexpression of Pyk2 causes constitutive STATc activation. Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc both in vitro and in vivo. This demonstration that a TKL directly activates a STAT has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predicted constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Here we show that the STATc-Pyk2 complex is formed constitutively by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.

Fig. 1.

Characterization of the pyk2⁻ strain. (A) Activation of STATc in parental Ax2 and pyk2⁻ cells. Parental Ax2 cells and pyk2⁻ cells were developed in shaken suspension for 4 h and then were treated with DIF-1 at 100 nM for the indicated times. Tyrosine phosphorylation of STATc was assayed by Western blot (IB) using phospho-STATc antibody CP22 (pSTATc, Top), and the same blot was reprobed with total-STATc antibody 7H3 (STATc, Middle). (Bottom) The combined data from several such experiments are quantified. (B) Nuclear translocation of STATc in parental and pyk2⁻ cells. Cells developed as in A were treated with DIF-1 at 100 nM for 3 min. The nuclear accumulation of STATc was assayed immunohistochemically using total STATc antibody. Ethanol-treated cells were used as controls, because ethanol is the vehicle for DIF-1. There are many fewer labeled nuclei in the null strain. (Scale bar: 10 μm.)

Fig. 2.

Characterization of a Pyk2 overexpressor strain. (A) STATc activation in parental and Pyk2-overexpressing cells. STATc activation was assayed in Ax2 cells and in Ax2 cells transformed with Myc-Pyk2 (Myc-Pyk2 OE cells) using DIF-1 at 100 nM, after 3 min of induction and as in Fig. 1A. When it is overexpressed in this way, Pyk2 interacts detectably with the monoclonal antibody used to monitor tyrosine phosphorylation of STATc. Hence we also confirmed the identity of STATc by analyzing the Myc-Pyk2 construct in a STATc-null background. (B) Nuclear localization of STATc in parental and Pyk2 OE cells. STATc nuclear translocation was assayed in cells developed in suspension induced with DIF-1, as in Fig. 1B, using Ax2 cells or Myc-Pyk2 OE cells. Note that the total STATc antibody, which is N-terminal epitope directed, was used to detect STATc; it does not cross-react with Pyk2. (Scale bar: 10 μm.)

Fig. 3.

Evidence that Pyk2 and STATc interact physically. (A and B) Co-IP of STATc and Pyk2. Ax2 cells or Myc-Pyk2 OE cells were developed in suspension as in Fig. 2A and were induced for the indicated times with DIF-1. (A) Lysates were immunoprecipitated using the 9E10 Myc antibody (+Ab) or were mock precipitated (−Ab). The precipitates were analyzed by Western blot using the total STATc antibody 7H3 and also with 9E10. The Myc antibody analysis confirms that the IP enriches for Myc-Pyk2. The 7H3 antibody analysis shows that STATc is coprecipitated only when Myc-Pyk2 is present and Myc antibody is added. DIF-1 treatment did not significantly affect the amount of STATc recovered. The same samples were reanalyzed with the 4G10 general phosphotyrosine antibody, and this analysis shows that Myc-Pyk2 is constitutively phosphorylated on tyrosine. (B) The reverse protocol to that in A was applied to Ax2 and Myc-Pyk2 OE lysates; i.e., the samples were precipitated with 7H3, and the Western blot was analyzed using 9E10 and, as a precipitation control, with 7H3. (C and D) GST pull-down assays of STATc and Pyk2. Ax2 cells and Myc-Pyk2 OE cells at 4 h of development in suspension were left untreated or were exposed to DIF-1 (100 nM) for 3 min. Cells then were lysed, and the extracts were subjected to pull-down assay. (C) For the Ax2 samples, GST-Pyk2 was used in the pull-down assay, and STATc was detected using the 7H3 total STATc antibody. (D) For the Myc-Pyk2 OE cells, GST-STATc was used in the pull-down assay, and the Myc-Pyk2 protein was detected using the 9E10 Myc antibody.

Fig. 4.

In vitro tyrosine phosphorylation of STATc by Pyk2. (A) Tyrosine phosphorylation of STATc using recombinant GST-Pyk2. GST-Pyk2 fusion proteins, produced in E. coli, were purified by glutathione affinity chromatography and were used in a kinase reaction with His-STATc or His-STATcR831A, a mutant form with an inactivating substitution in the SH2 domain, as substrate. The reaction products were assayed for STATc tyrosine phosphorylation by Western blotting using phospho-STATc antibody. (B) Tyrosine phosphorylation of STATc was assayed immunologically using immunopurified Myc-Pyk2. Ax2 cells transformed with Myc-Pyk2 (Myc-Pyk2 OE cells) or with its kinase-dead form, Myc-Pyk2K880A (Myc-Pyk2K880A OE cells) were lysed at 4 h of suspension development, and enzyme immunopurified from them was assayed for STATc TK activity. In addition to the substrate, GST-STATcΔ, a major low-mobility species, Myc-Pyk2, is autophosphorylated. Neither protein species is observed when the kinase-dead K880A form of Myc-Pyk2 is used as enzyme. (C) Time course of the activation of STATc by DIF-1. A time course of the DIF-1 induction of STATc tyrosine phosphorylation activity by Myc-Pyk2 was generated as in B.

Fig. 5.

Determination of the role of Pyk2 autophosphorylation. (A) Analysis of the interaction of kinase-dead Myc-Pyk2 with STATc by co-IP assay. Myc-Pyk2 OE or Myc-Pyk2K880A OE cells were induced with DIF-1, and then Pyk2 was immunoprecipitated with 9E10 Myc antibody and analyzed for STATc binding using the 7H3 total STATc antibody as in Fig. 3A. As a loading control, the blot was reprobed for Myc-Pyk2 using 9E10 Myc antibody. The same samples were reanalyzed with the 4G10 general phosphotyrosine antibody; this analysis shows that Myc-Pyk2 is constitutively phosphorylated on tyrosine. Presumably because of their difference in phosphorylation status, the parental and kinase-dead forms of Myc-Pyk2 display a small difference in mobility indicated by a double arrowhead. (B) Analysis of the interaction of wild-type and kinase-dead GST-Pyk2 with STATc. Ax2 cells were developed for 4 h and then were left untreated or were exposed to DIF-1 as in Fig. 4B. Cells were lysed, and the extracts were subjected to pull-down assay using GST-Pyk2 or GST-Pyk2K880A (its kinase-dead form) and assaying STATc by Western blot as in Fig. 3C. The blot was reprobed with a GST antibody as a loading control. (C) Determination of the phosphatase sensitivity of GST-Pyk2 binding to STATc. GST-Pyk2 was bound to glutathione beads, and the beads either were left untreated or were digested with the TC PTP tyrosine phosphatase. A parallel reaction was performed on GST-Pyk2 beads in the presence of sodium orthovanadate, an inhibitor of the enzyme. The various beads were used in pull down of STATc in extracts from cells left untreated or induced with DIF-1 and were assayed as in Fig. 3B. The blot was reprobed with a GST antibody as a loading control, and the same samples were reanalyzed with the 4G10 general phosphotyrosine antibody.

Fig. 6.

Determination of the role of the STATc SH2 domain. (A) Pull-down analysis of Dictyostelium-expressed Myc-Pyk2 by mutant forms of STATc. Myc-Pyk2 OE cell lysates from control cells or cells induced with DIF-1 were subjected to pull-down assay using GST-STATc or its R831A- and Y922F-mutant forms as in Fig. 3B. The blot was reprobed with a GST antibody as a loading control. (B) Pull-down analysis of recombinant Pyk2, STATc, and its R831A-mutant form. GST-Pyk2, His-STATc, and His-STATcR831A, all expressed in E. coli, were subjected to affinity chromatography using their respective tags. The GST-Pyk2 fusion protein was attached to the glutathione beads, and aliquots were treated with TC PTP with or without sodium orthovanadate, as in Fig. 5C. The His-tagged STATc proteins were used in binding to the GST-Pyk2/glutathione beads as indicated. The recovered STATc protein was assayed by Western blot using a His antibody, or a GST antibody as indicated.

Fig. P1.

A scheme for the regulation of STATc tyrosine phosphorylation by Pyk2. The following sequence of events is proposed: (1) Pyk2 autophosphorylates on one or more tyrosine residues. (2) STATc binds to a tyrosine residue on Pyk2 via the SH2 domain of STATc. (3) Pyk2 transiently phosphorylates Tyr922 of STATc but, in a competing reaction, the PTP3 protein tyrosine phosphatase catalyzes dephosphorylation at the same site (ref. 5). We assume that there is an equilibrium between the STATc bound to Pyk2 and the free STATc and that the relative kinase and phosphatase activity levels determine the proportion in each pool. (4) Upon exposure to DIF-1, a small organic molecule produced by the cells, PTP3 becomes phosphorylated on two serine residues, S448 and S747, and thus is inhibited. This inhibition results in net tyrosine phosphorylation, i.e., activation, of STATc. (5) Activated STATc dissociates from Pyk2 and dimerizes.