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. S H2 domains form part of a paradigmatic "writer/reader/ eraser" control module (1, 2) in which the writers are tyrosine kinases (TKs), the readers are SH2 domains, and the erasers are protein tyrosine phosphatases (PTPs). In principle no one element of such a three-component system can function usefully independently of the other two. So how could such a three-component system have evolved? Here the fungi and the amoebozoan Dictyostelium have been informative.Metazoa possess large numbers of TKs, but the only unicellular organisms known to possess them are choanoflagellates, unicellular close relatives of the Metazoa (3, 4). Although neither Dictyostelium nor fungi possess TKs, they do have PTPs, thus suggesting that tyrosine phosphorylation arose in a common ancestor of fungi and Amoebozoa, mediated perhaps by dualspecificity kinases and regulated by dephosphorylation via PTPs (2). There are no phosphotyrosine-binding SH2 domains in fungi, but there is a sequence-related domain within the SPT6 protein that binds phospho-serine and that may have been ancestral to modern SH2 domains (5). In the fungi, therefore, phosphorylation of tyrosine appears to be used solely as a direct modulator of enzymatic function, but in Dictyoste...

Analysis of DrkA kinase’s role in STATa activation

“…It had been a mystery as to which kinases (TKLs) specifically activate Dictyostelium STATs. Recently, however, two TKL kinases that phosphorylate and activate STATc were identified (Araki, Kawata, & Williams, ; Araki et al, ). STATc is activated by either hyperosmotic stress or ligand stimulation (Araki et al, ; Fukuzawa, Araki, Adrian, & Williams, ; Na, Tunggal, & Eichinger, ), where the ligand is differentiation‐inducing factor‐1 (DIF‐1), a chlorinated polyketide necessary to induce prestalk‐specific gene expression (Williams, ).…”

“…STATc is activated by either hyperosmotic stress or ligand stimulation (Araki et al, ; Fukuzawa, Araki, Adrian, & Williams, ; Na, Tunggal, & Eichinger, ), where the ligand is differentiation‐inducing factor‐1 (DIF‐1), a chlorinated polyketide necessary to induce prestalk‐specific gene expression (Williams, ). The TKL involved in DIF‐1‐induced STATc activation is Pyk2 (Araki et al, ), whereas Pyk2 and Pyk3 are the TKLs that activate STATc in response to hyperosmotic stress (Araki et al, ). Protein tyrosine phosphatase PTP3 has been showed to control the level of STATc phosphorylation.…”

“…As a consequence of DIF‐1 induction or hyperosmotic stress, however, PTP3 is phosphorylated at Ser448 and Ser747, which results in the reduction of PTP3 activity and, thereby, increased STATc phosphorylation on Tyr922 (Araki, Langenick, Gamper, Firtel, & Williams, ). Because complex formation between STATc and Pyk2 is constitutive, Pyk2 functions as a constitutively active STATc kinase, whereas PTP3 serves as the principal regulator of STATc activation (Araki et al, ).…”

Evolution of the JAK-STAT pathway

“…Of course, each of the components would have necessarily participated in other processes prior to their recruitment into the canonical JAK-STAT pathway. Indeed, the legacy of this is evident in some of the non-canonical roles played by the pathway components in alternate signaling paradigms, such as downstream of receptor tyrosine kinases 28 , 31 , 52 , 54 , 55 …”

The Dictyostelium prestalk inducer DIF-1 directs phosphorylation of a bZIP transcription factor

“…There, Pyk2, a Tyrosine Kinase Like enzyme, phosphorylates STATc on a tyrosine residue, Y922, located close to the C terminus (Araki et al, 2012). This activation is constitutive and regulation by DIF-1 is exerted via DIF's inhibitory effect on PTP3; a protein tyrosine phosphatase that acts to dephosphorylate Y922 (Araki et al, 2008).…”