Implications of an antiparallel dimeric structure of nonphosphorylated STAT1 for the activation–inactivation cycle

Abstract

IFN-␥ treatment of cells leads to tyrosine phosphorylation of signal transducer and activator of transcription (STAT) 1 followed by dimerization through a reciprocal Src homology 2-phosphotyrosine interaction near the -COOH end of each monomer, forming a parallel structure that accumulates in the nucleus to drive transcription. Prompt dephosphorylation and return to the cytoplasm completes the activation-inactivation cycle. Nonphosphorylated STATs dimerize, and a previously described interface between N-terminal domain (ND) dimers has been implicated in this dimerization. A new crystal structure of nonphosphorylated STAT1 containing the ND dimer has two possible configurations for the body of STAT1, one of which is antiparallel. In this antiparallel structure, the Src homology 2 domains are at opposite ends of the dimer, with the coiled:coil domain of one monomer interacting reciprocally with the DNA-binding domain of its partner. Here, we find that mutations in either the coiled:coil͞DNA-binding domain interface or the ND dimer interface block dimerization of nonphosphorylated molecules and cause a resistance to dephosphorylation in vivo and resistance to a tyrosine phosphatase in vitro. We conclude that a parallel STAT1 phosphodimer not bound to DNA most likely undergoes a conformational rearrangement (parallel to antiparallel) to present the phosphotyrosine efficiently for dephosphorylation.dephosphorylation ͉ structural rearrangement S ignal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that can be activated by a variety of tyrosine kinases in response to many different cytokine, growth factor, and peptide ligands binding to their respective cell surface receptors. Accumulation in the nucleus of tyrosine phosphorylated STAT dimers is followed by DNA binding, activation of target gene transcription, dephosphorylation, and return to the cytoplasm (1). The crystal structure of the phosphodimer core (amino acids 130-712), bound to DNA, showed a reciprocal phosphotyrosine (pY)-Src homology 2 (SH2) interaction at one end of a parallel dimer (2), with the DNA separating the monomers along the long axis of the monomers. Mao et al. (3) have now described a dimeric structure of nonphosphorylated STAT1, including the core (amino acids 130-712), as well as the core plus the N-terminal domain (ND) (amino acids 1-683). (The terminal 29 aa, 684-712, in the core were unstructured and omitted from study.) The body of each of the monomers in the nonphosphorylated structure is identical to the monomers in the previously reported phosphorylated dimer (2). However, the monomers in the nonphosphorylated core structure are arranged differently in space. In contrast to the dimeric, parallel structure of the phosphorylated STAT1, the SH2 domains in the nonphosphorylated core structure project from the opposite ends of an antiparallel dimer with a dimeric interface formed by reciprocal contacts between the coiled:coil (CC) and DNA-binding domains (DBD) of the monomers. In the crystal...

A phosphorylation-acetylation switch regulates STAT1 signaling

“…The ''pocket'' residues Q 340 , Q 408 , and G 384 are required for spatial reorientation of a parallel to an anti-parallel STAT1 dimer presenting the pY 701 to TCP45 (Zhong et al 2005;Mertens et al 2006). It appears feasible that after initial activation, and once acetylated in the nucleus by CBP, STAT1 is stabilized in its anti-parallel structure providing access for TCP45.…”

Phosphorylation–acetylation switch in the regulation of STAT1 signaling

“…Remarkably, dimerization of STAT1 is not necessary for the initiation of IFN-dependent signaling [33,34]. Moreover, positive and negative transcriptional control can also be exerted by unphosphorylated STAT1 via mechanisms distinct from those used by phosphorylated STAT1 [35][36][37][38].…”

“…A radical transition from parallel to antiparallel conformation, which allows the presentation of the phosphorylated tyrosine residues of STAT1 dimers to TCP45, has been suggested [34,61]. The existence of multiple layers of temporal and spatial regulation of cytokine-induced signaling allows precise control of genes important for metazoan growth, immune defense and development via STAT1.…”

A partial form of recessive STAT1 deficiency in humans

“…IFN-γ stimulation leads to activation of the associated Janus kinases JAK1 and JAK2, which phosphorylate the tetrameric IFN-γ receptor, creating a docking site for 2 latent STAT1 molecules. These molecules are phosphorylated on their Tyr701 residues and are released into the cytosol as phosphorylated active STAT1 homodimers, forming gamma-activating factor (GAF) (1,6,7). GAF is translocated to the nucleus (8), where it binds to gamma-activating sequences (GASs) in the promoters of target genes and activates transcription through the transactivator domain of STAT1 (1).…”