STAT proteins are a family of latent transcription factors that mediate the response to various cytokines and growth factors. Upon stimulation by cytokines, STAT proteins are recruited to the receptors via their SH2 domains, phosphorylated on a specific tyrosine, dimerized, and translocated into the nucleus, where they bind specific DNA sequences and activate the target gene transcription. STATs share highly conserved structures, including an N-domain, a coiled-coil domain, a DNA-binding domain, a linker domain, and an SH2 domain. To investigate the role of the coiled-coil domain, we performed a systematic deletion analysis of the N-domain and each of the ␣-helices and mutagenesis of conserved residues in the coiled-coil region of Stat3. Our results indicate that the coiled-coil domain is essential for Stat3 recruitment to the receptor and the subsequent tyrosine phosphorylation and tyrosine phosphorylation-dependent activities, such as dimer formation, nuclear translocation, and DNA binding, stimulated by epidermal growth factor (EGF) or interleukin-6 (IL-6). Single mutation of Asp170 or, to a lesser extent, Lys177 in ␣-helix 1 diminishes both receptor binding and tyrosine phosphorylation. Furthermore, the Asp170 mutant retains its ability to bind to DNA when phosphorylated on Tyr705 by Src kinase in vitro, implying a functional SH2 domain. Finally, we demonstrate a direct binding of Stat3 to the receptor. Taken together, our data reveal a novel role for the coiled-coil domain that regulates the early events in Stat3 activation and function.Growth factors and cytokines regulate cell growth and differentiation by triggering various intracellular signaling pathways that lead to gene expression. JAK-STAT is a key pathway that mediates cellular responses to a variety of cytokines (reviewed in references 11, 20, and 30). As the name suggests, STATs (signal transducers and activators of transcription) represent a family of proteins with dual functions that transduce the signal in the cytoplasm and activate gene expression in the nucleus. Seven STATs with different functions have been identified so far in mammalian cells, and over 40 different polypeptides are known to activate one or more STATs (reviewed in reference 10). Among them, Stat3 was identified both as an acute-phase response factor activated by interleukin-6 (IL-6) in mouse liver and by homology to Stat1 (2, 46). Stat3 is also activated by other members of the IL-6 family, such as IL-11, ciliary neurotrophic factor, oncostatin M, and leukemia-inhibitory factor, which share the common transducing gp130 receptor subunit (2, 25, 33). IL-6, as a pleiotropic cytokine, exhibits various functions in immune response, hematopoiesis, and neuronal differentiation (37).The model of activation of Stat3 by IL-6 has been established. Binding of IL-6 to its receptor gp80 (subunit ␣) induces homodimerization of gp130 (subunit ␤) and phosphorylation of the gp130-associated Janus kinases (JAKs). JAKs phosphorylate the tyrosine residues on gp130 that serve as docking sites fo...

Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is a Cdc42-binding serine/threonine kinase with multiple functional domains. We had previously shown MRCK␣ to be implicated in Cdc42-mediated peripheral actin formation and neurite outgrowth in HeLa and PC12 cells, respectively. Here we demonstrate that native MRCK exists in high-molecular-weight complexes. We further show that the three independent coiled-coil (CC) domains and the N-terminal region preceding the kinase domain are responsible for intermolecular interactions leading to MRCK␣ multimerization. N terminus-mediated dimerization and consequent transautophosphorylation are critical processes regulating MRCK␣ catalytic activities. A region containing the two distal CC domains (CC2 and CC3; residues 658 to 930) was found to interact intramolecularly with the kinase domain and negatively regulates its activity. Its deletion also resulted in an active kinase, confirming a negative autoregulatory role. We provide evidence that the N terminus-mediated dimerization and activation of MRCK and the negative autoregulatory kinase-distal CC interaction are two mutually exclusive events that tightly regulate the catalytic state of the kinase. Disruption of this interaction by a mutant kinase domain resulted in increased kinase activity. MRCK kinase activity was also elevated when cells were treated with phorbol ester, which can interact directly with a cysteine-rich domain next to the distal CC domain. We therefore suggest that binding of phorbol ester to MRCK releases its autoinhibition, allowing N-terminal dimerization and subsequent kinase activation.

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We recently showed that BNIP-2 is a putative substrate of the fibroblast growth factor receptor tyrosine kinase and it possesses GTPase-activating activity toward the small GTPase, Cdc42. The carboxyl terminus of BNIP-2 shares high homology to the non-catalytic domain of Cdc42GAP, termed BCH (for BNIP-2 and Cdc42GAP homology) domain. Despite the lack of obvious homology to any known catalytic domains of GTPase-activating proteins (GAPs), the BCH domain of BNIP-2 bound Cdc42 and stimulated the GTPase activity via a novel arginine-patch motif similar to that employed by one contributing partner in a Cdc42 homodimer. In contrast, the BCH domain of Cdc42GAP, although it can bind Cdc42, is catalytically inactive. This raises the possibility that these domains might have other roles in the cell. Using glutathione S-transferase recombinant proteins, immunoprecipitation studies, and yeast two-hybrid assays, it was found that BNIP-2 and Cdc42GAP could form homo and hetero complexes via their conserved BCH domains. Molecular modeling of the BNIP-2 BCH homodimer complex and subsequent deletion mutagenesis helped to identify the region 217 RRKMP221 as the major BCH interaction site within BNIP-2. In comparison, deletion of either the argininepatch 235 RRLRK 239 (necessary for GAP activity) or region 288 EYV 290 (a Cdc42 binding sequence) had no effect on BCH-BCH interaction. Extensive data base searches showed that the BCH domain is highly conserved across species. The results suggest that BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain that could potentially determine and/or modify the physiological roles of these molecules.We recently identified BNIP-2, a previously cloned Bcl-2 and adenovirus E1B-interacting protein (1), as a putative substrate of the fibroblast growth factor receptor tyrosine kinase. When not tyrosine-phosphorylated BNIP-2 can bind to two cellular targets: Cdc42, a small GTPase and its regulator, Cdc42GAP but this binding is abrogated upon its tyrosine phosphorylation (2).Cdc42 is a member of the Rho subfamily of GTPases demonstrated to be involved in various aspects of cytoskeletal organization, regulation of the transcription of certain target genes, and the control of aspects of cell cycle progression (3-12). GTPases cycle between the inactive, GDP-bound form and the active GTP-bound form. The equilibrium between these two states is controlled at least by two major classes of regulators, the guanine nucleotide exchange factors and the GTPase-activating proteins (GAPs) 1 (13-15). The guanine nucleotide exchange factors catalyze the exchange of GDP on the inactive GTPase for GTP, which results in enhanced activity of the target protein. The GAPs enhance rates of GTP hydrolysis to GDP mainly by contributing catalytic arginine residues to their substrate target in trans, or by stabilizing the conformation of the inherent GTPases (15-24).Despite lacking obvious sequence homology to the canonical catalytic domain of GAP proteins, BNIP-2 was shown to possess a GAP...