The constituent polypeptides of the interleukin-17 family form six different homodimeric cytokines (IL-17A-F) and the heterodimeric IL-17A/F. Their interactions with IL-17 receptors A-E (IL-17RA-E) mediate host defenses while also contributing to inflammatory and autoimmune responses. IL-17A and IL-17F both preferentially engage a receptor complex containing one molecule of IL-17RA and one molecule of IL-17RC. More generally, IL-17RA appears to be a shared receptor that pairs with other members of its family to allow signaling of different IL-17 cytokines. Here we report crystal structures of homodimeric IL-17A and its complex with IL-17RA. Binding to IL-17RA at one side of the IL-17A molecule induces a conformational change in the second, symmetry-related receptor site of IL-17A. This change favors, and is sufficient to account for, the selection of a different receptor polypeptide to complete the cytokine-receptor complex. The structural results are supported by biophysical studies with IL-17A variants produced by site-directed mutagenesis.

SHP-1 is a cytosolic protein-tyrosine phosphatase that behaves as a negative regulator in eukaryotic cellular signaling pathways. To understand its regulatory mechanism, we have determined the crystal structure of the C-terminal truncated human SHP-1 in the inactive conformation at 2.8-Å resolution and refined the structure to a crystallographic R-factor of 24.0%. The three-dimensional structure shows that the ligand-free SHP-1 has an auto-inhibited conformation. Its N-SH2 domain blocks the catalytic domain and keeps the enzyme in the inactive conformation, which supports that the phosphatase activity of SHP-1 is primarily regulated by the N-SH2 domain. In addition, the C-SH2 domain of SHP-1 has a different orientation from and is more flexible than that of SHP-2, which enables us to propose an enzymatic activation mechanism in which the C-SH2 domains of SHPs could be involved in searching for phosphotyrosine activators.Tyrosine phosphorylation is a key mechanism for regulating eukaryotic cellular signaling pathways. The protein tyrosine phosphorylation level is precisely regulated by two types of enzymes: protein-tyrosine kinases (PTKs) 1 and protein-tyrosine phosphatases (PTPs), in which PTPs act to counter-balance the process through dephosphorylation of the phosphorylated tyrosines (1, 2). PTPs can be divided into two groups, receptor protein-tyrosine phosphatases and cytosolic proteintyrosine phosphatases. The SH2 domain-containing PTPs, SHP-1 and SHP-2, are both cytosolic PTPs and share many structural and regulatory features. They both have two tandem SH2 domains at the N terminus followed by a single catalytic domain and an inhibitory C-terminal tail. However, irrespective of similar structural and regulatory characteristics, these two enzymes have different biological function in vivo.Different from SHP-2, which is expressed in all kinds of tissues, SHP-1 is predominantly expressed in hematopoietic and epithelial cells and behaves mainly as a negative regulator of signaling pathways in lymphocytes (1, 2). SHP-1 is dormant in the cytosol, with its phosphatase activity inhibited by both the SH2 domains and the C-terminal tail (1,(3)(4)(5). In response to an activation signal, SHP-1 is recruited to membrane-bound inhibitory receptors via the binding of its SH2 domains to the tyrosine-phosphorylated immunoreceptor tyrosine-based inhibitory motif within the cytoplasmic domain of a receptor (6 -8). During this process, SHP-1 undergoes a structural rearrangement, exposes its active site, and binds to the downstream substrates, thereby dephosphorylating the substrates to turn off the cellular signals.SHP-1 also presents in several types of non-hematopoietic cells (9 -12). Overexpression of a catalytically inactive SHP-1 mutant in these cells strongly suppressed mitogen-activated pathways, reducing signal transduction and activation of transcription; these findings demonstrate that SHP-1 has a positive effect on mitogenic signaling in these non-hematopoietic cells (10, 11). Thus, SHP-1 probably has both the nega...

The Phox homology (PX) domain has recently been reported to bind to phosphoinositides, and some PX domains can localize to endosomes in vivo. Here we show data to support the conclusion that the p40 phox PX domain binds to phosphatidylinositol 3-phosphate specifically in vitro and localizes to endosomes in intact cells. In addition, its Y59A/L65Q mutant, which has decreased affinity for phosphatidylinositol 3-phosphate in vitro, fails to target EGFP-p40-PX to endosomes. However, unlike published results, we find that the p47 phox PX domain weakly binds to many phosphoinositides in vitro showing slightly higher affinity for phosphatidylinositol 3,4,5-trisphosphate. Moreover, we show for the first time that upon insulin-like growth factor-1 stimulation of COS cells, the p47 phox PX domain is localized to the plasma membrane, and this subcellular localization is dependent on PI 3-kinase activity. Unexpectedly, its R42Q mutant that loses in vitro phosphoinositide-binding ability can still target EGFP-p47-PX to the plasma membrane. Our data suggest that the translocation of p47 phox PX domain to the plasma membrane does involve 3-phosphoinositide(s) in the process, but the phosphoinositide-binding of p47 phox PX domain is not sufficient to recruit it to the plasma membrane. Therefore, the p40 phox and p47 phox PX domains can target subcellular membranes via direct or indirect recruitment by phosphoinositides, while both are under the control of phosphatidylinositol 3-kinase activity. phox , specifically interact with PI(3)P. They can also target those proteins to endosomes in vivo (2-5, 7, 8). In addition, 14 out of 15 PX domains from yeast bind to PI(3)P with different affinity (40). However, the PX domain of CPK PI 3-kinase interacts with PI(4,5)P 2 (6), while the p47 phox PX domain has been reported to preferentially interact with PI(3,4)P 2 (5). Moreover, the PX domain of p47 phox shows binding to its C-terminal SH3 domain (9).NADPH oxidase, consisting of both membrane-associated subunits and cytosolic subunits, plays a very important role in the immune defense system of neutrophils and other phagocytic cells (for a recent review, see Ref. 12). Defects in those subunits will result in dysfunction of the enzyme and clinically will cause chronic granulomatous disease. Patients with chronic granulomatous disease suffer severe, recurrent bacterial and fungal infections. In the resting state, the subunits of NADPH oxidase are differentially localized to the membrane (gp91 phox and p22 phox ) and to the cytoplasm (p40 phox , p67 phox , and p47 phox ). In response to invasive microorganisms, the cytosolic subunits of NADPH oxidase translocate to the membrane and fuse with the membrane-bound subunits to form an activated enzyme complex. The core part of the active enzyme consists of the cytosolic subunit p67 phox , a GTPase Rac1/2, the membrane subunits gp91 phox and p22 phox , while p47 phox is required for the assembly of the active NADPH oxidase complex acting as an adaptor for p67 phox (13)(14)(15), and the role of p...

SHP-1 belongs to the family of non-receptor protein tyrosine phosphatases (PTPs) and generally acts as a negative regulator in a variety of cellular signaling pathways. Previously the crystal structures of the tail-truncated SHP-1 and SHP-2 revealed an autoinhibitory conformation. To understand the regulatory mechanism of SHP-1, we have determined the crystal structure of the full length SHP-1 at 3.1 Å. Although the tail was disordered in current structure, the huge conformational rearrangement of the N-SH2 domain and the incorporation of sulfate ions into the ligand-binding site of each domain indicate that the SHP-1 is in the open conformation. The N-SH2 domain in current structure is shifted away from the active site of the PTP domain to the other side of the C-SH2 domain, resulting in exposure of the active site. Meanwhile, the C-SH2 domain is twisted anticlockwise by about 110°. In addition, a set of new interactions between two SH2 domains and between the N-SH2 and the catalytic domains is identified, which could be responsible for the stabilization of SHP-1 in the open conformation. Based on the structural comparison, a model for the activation of SHP-1 is proposed.

The recruitment of specific cytosolic proteins to intracellular membranes through binding phosphorylated derivatives of phosphatidylinositol (PtdIns) controls such processes as endocytosis, regulated exocytosis, cytoskeletal organization, and cell signaling. Protein modules such as FVYE domains and PH domains that bind specifically to PtdIns 3-phosphate (PtdIns-3-P) and polyphosphoinositides, respectively, can direct such membrane targeting. Here we show that two representative Phox homology (PX) domains selectively bind to specific phosphatidylinositol phosphates. The PX domain of Vam7p selectively binds PtdIns-3-P, while the PX domain of the CPK PI-3 kinase selectively binds PtdIns-4,5-P(2). In contrast, the PX domain of Vps5p displays no binding to any PtdInsPs that were tested. In addition, the double mutant (Y42A/L48Q) of the PX domain of Vam7p, reported to cause vacuolar trafficking defects in yeast, has a dramatically decreased level of binding to PtdIns-3-P. These data reveal that the membrane targeting function of the Vam7p PX domain is based on its ability to associate with PtdIns-3-P, analogous to the function of FYVE domains.