Signalling through SH2 and SH3 domains

In 1986, Pawson's group recognized a region of homology between two oncogenic tyrosine kinases that lay outside the catalytic domain. They termed this the Src homology 2, or SH2, domain. In the ensuing years, SH2 domains have been found in an impressive variety of proteins, as has a second region of homology, inevitably termed SH3. These domains appear to mediate controlled protein-protein interactions. Many proteins that contain SH2 and SH3 domains are involved in signal transduction, suggesting a new paradigm for regulation of intracellular signalling pathways.     

Diversity in peptide recognition by the SH2 domain of SH2B1

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein‐protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate‐1 and ?2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C‐terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high‐resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand‐binding interface of SH2B1 with high specificity.     

The SH2 Domain Interaction Landscape

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Highlights? The recognition specificity of 70 SH2 domains is probed ? Recognition specificity diverges faster than sequence ? PepspotDB is a database of protein interactions mediated by SH2 domains     

Hepatic SH2B1 and SH2B2 Regulate Liver Lipid Metabolism and VLDL Secretion in Mice

SH2B1 is an SH2 and PH domain-containing adaptor protein. Genetic deletion of SH2B1 results in obesity, type 2 diabetes, and fatty liver diseases in mice. Mutations in SH2B1 are linked to obesity in humans. SH2B1 in the brain controls energy balance and body weight at least in part by enhancing leptin sensitivity in the hypothalamus. SH2B1 in peripheral tissues also regulates glucose and lipid metabolism, presumably by enhancing insulin sensitivity in peripheral metabolically-active tissues. However, the function of SH2B1 in individual peripheral tissues is unknown. Here we generated and metabolically characterized hepatocyte-specific SH2B1 knockout (HKO) mice. Blood glucose and plasma insulin levels, glucose tolerance, and insulin tolerance were similar between HKO, albumin-Cre, and SH2B1^f/f mice fed either a normal chow diet or a high fat diet (HFD). Adult-onset deletion of SH2B1 in the liver either alone or in combination with whole body SH2B2 knockout also did not exacerbate HFD-induced insulin resistance and glucose intolerance. Adult-onset, but not embryonic, deletion of SH2B1 in the liver attenuated HFD-induced hepatic steatosis. In agreement, adult-onset deletion of hepatic SH2B1 decreased the expression of diacylglycerol acyltransferase-2 (DGAT2) and increased the expression of adipose triglyceride lipase (ATGL). Furthermore, deletion of liver SH2B1 in SH2B2 null mice attenuated very low-density lipoprotein (VLDL) secretion. These data indicate that hepatic SH2B1 is not required for the maintenance of normal insulin sensitivity and glucose metabolism; however, it regulates liver triacylglycerol synthesis, lipolysis, and VLDL secretion.     

Membrane-targeting of signalling molecules by SH2/SH3 domain-containing adaptor proteins

SH2/SH3 domain-containing adaptor proteins play a critical role in regulating tyrosine kinase signalling pathways. The major function of these adaptors, such as Grb2, Nck, and Crk, is to recruit proline-rich effector molecules to tyrosine-phosphorylated kinases or their substrates. In recent years dozens of novel proteins have emerged that are capable of associating with the SH2 and the SH3 domains of adaptors. In this review, the author attempts to summarise these novel binding partners of Grb2, Nck, and Crk, and to discuss current controversies regarding function and regulation of protein multicomplexes held together by SH2/SH3 adaptor molecules at the plasma membrane.     

Membrane-targeting of signalling molecules by SH2/SH3 domain-containing adaptor proteins.

SH2/SH3 domain-containing adaptor proteins play a critical role in regulating tyrosine kinase signalling pathways. The major function of these adaptors, such as Grb2, Nck, and Crk, is to recruit proline-rich effector molecules to tyrosine-phosphorylated kinases or their substrates. In recent years dozens of novel proteins have emerged that are capable of associating with the SH2 and the SH3 domains of adaptors. In this review, the author attempts to summarise these novel binding partners of Grb2, Nck, and Crk, and to discuss current controversies regarding function and regulation of protein multicomplexes held together by SH2/SH3 adaptor molecules at the plasma membrane.     

Differential binding to and regulation of JAK2 by the SH2 domain and N-terminal region of SH2-bbeta

SH2-Bbeta has been shown to bind via its SH2 (Src homology 2) domain to tyrosyl-phosphorylated JAK2 and strongly activate JAK2. In this study, we demonstrate the existence of an additional binding site(s) for JAK2 within the N-terminal region of SH2-Bbeta (amino acids 1 to 555) and the ability of this region of SH2-B to inhibit JAK2. Four lines of evidence support the existence of this additional binding site(s). In a glutathione S-transferase pull-down assay, wild-type SH2-Bbeta and SH2-Bbeta(R555E) with a defective SH2 domain bind to both tyrosyl-phosphorylated JAK2 from growth hormone (GH)-treated cells and non-tyrosyl-phosphorylated JAK2 from control cells, whereas the SH2 domain of SH2-Bbeta binds only to tyrosyl-phosphorylated JAK2 from GH-treated cells. Similarly, JAK2 is present in alphaSH2-B immunoprecipitates in the absence and presence of GH, with GH substantially increasing the coprecipitation of JAK2 with SH2-B. When coexpressed in COS cells, SH2-Bbeta coimmunoprecipitates not only wild-type, tyrosyl-phosphorylated JAK2 but also kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E), although to a lesser extent. DeltaC555 (amino acids 1 to 555 of SH2-Bbeta) that lacks most of the SH2 domain binds similarly to wild-type JAK2 and kinase-inactive JAK2(K882E). Experiments using a series of N- and C-terminally truncated SH2-Bbeta constructs indicate that the pleckstrin homology (PH) domain (amino acids 269 to 410) and amino acids 410 to 555 are necessary for maximal binding of SH2-Bbeta to inactive JAK2, but neither region alone is sufficient for maximal binding. The SH2 domain of SH2-Bbeta is necessary and sufficient for the stimulatory effect of SH2-Bbeta on JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B. In contrast, DeltaC555 lacking the SH2 domain, and to a lesser extent the PH domain alone, inhibits JAK2. DeltaC555 also blocks JAK2-mediated tyrosyl phosphorylation of Stat5B in COS cells and GH-stimulated nuclear accumulation of Stat5B in 3T3-F442A cells. These data indicate that in addition to the SH2 domain, SH2-Bbeta has one or more lower-affinity binding sites for JAK2 within amino acids 269 to 555. The interaction via this site(s) in SH2-B with inactive JAK2 seems likely to increase the local concentration of SH2-Bbeta around JAK2, thereby facilitating binding of the SH2 domain to ligand-activated JAK2. This would result in a more rapid and robust cellular response to hormones and cytokines that activate JAK2. This interaction between inactive JAK2 and SH2-B may also help prevent abnormal activation of JAK2.     

High-throughput phosphotyrosine profiling using SH2 domains

Protein tyrosine phosphorylation controls many aspects of signaling in multicellular organisms. One of the major consequences of tyrosine phosphorylation is the creation of binding sites for proteins containing Src homology 2 (SH2) domains. To profile the global tyrosine phosphorylation state of the cell, we have developed proteomic binding assays encompassing nearly the full complement of human SH2 domains. Here we provide a global view of SH2 domain binding to cellular proteins based on large-scale far-western analyses. We also use reverse-phase protein arrays to generate comprehensive, quantitative SH2 binding profiles for phosphopeptides, recombinant proteins, and entire proteomes. As an example, we profiled the adhesion-dependent SH2 binding interactions in fibroblasts and identified specific focal adhesion complex proteins whose tyrosine phosphorylation and binding to SH2 domains are modulated by adhesion. These results demonstrate that high-throughput comprehensive SH2 profiling provides valuable mechanistic insights into tyrosine kinase signaling pathways.     

The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.     

Solid-phase binding assays of peptides using EGFP-Src SH2 domain fusion protein and biotinylated Src SH2 domain

Two solid-phase binding assays were designed and evaluated for their potential use in comparing the affinity of peptides to the Src SH2 domain. Resin beads attached to peptides were incubated with the enhanced green fluorescence protein(EGFP)-Src SH2 domain fusion protein or the biotinylated Src SH2 domain and extensively washed. The beads-attached tetrapeptides with high affinities to the EGFP-Src SH2 domain showed more fluorescence intensity than those beads containing tetrapeptides with weak binding affinities, as shown by fluorescence microscopy and fluorescence imaging system. Only the beads attached to pYEEI produced a dark purple color on incubation of the beads, respectively, with the biotinylated Src kinases SH2 domain, alkaline phosphatase-coupled streptavidin, and BCIP/NBT. These solid-phase binding assays may have potential applications for the screening of peptides for the Src kinases SH2 domains.     

SHIP2 (SH2 Domain-containing Inositol Phosphatase 2) SH2 Domain Negatively Controls SHIP2 Monoubiquitination in Response to Epidermal Growth Factor

The SH2 domain containing inositol 5-phosphatase SHIP2 contains several interacting domains that are important for scaffolding properties. We and others have previously reported that SHIP2 interacts with the E3 ubiquitin ligase c-Cbl. Here, we identified human SHIP2 monoubiquitination on lysine 315. SHIP2 could also be polyubiquitinated but was not degraded by the 26 S proteasome. Furthermore, we identified a ubiquitin-interacting motif at the C-terminal end of SHIP2 that confers ubiquitin binding capacity. However, this ubiquitin-interacting motif is dispensable for its monoubiquitination. We showed that neither c-Cbl nor Nedd4-1 play the role of ubiquitin ligase for SHIP2. Strikingly, monoubiquitination of the ΔSH2-SHIP2 mutant (lacking the N-terminal SH2 domain) is strongly increased, suggesting an intrinsic inhibitory effect of the SHIP2 SH2 domain on its monoubiquitination. Moreover, SHIP2 monoubiquitination was increased upon 30 min of epidermal growth factor stimulation. This correlates with the loss of interaction between the SHIP2 SH2 domain and c-Cbl. In this model, c-Cbl could mask the monoubiquitination site and thereby prevent SHIP2 monoubiquitination. The present study thus reveals an unexpected and novel role of SHIP2 SH2 domain in the regulation of its newly identified monoubiquitination.     

CD150 association with either the SH2-containing inositol phosphatase or the SH2-containing protein tyrosine phosphatase is regulated by the adaptor protein SH2D1A

CD150 (SLAM/IPO-3) is a cell surface receptor that, like the B cell receptor, CD40, and CD95, can transmit positive or negative signals. CD150 can associate with the SH2-containing inositol phosphatase (SHIP), the SH2-containing protein tyrosine phosphatase (SHP-2), and the adaptor protein SH2 domain protein 1A (SH2D1A/DSHP/SAP, also called Duncan's disease SH2-protein (DSHP) or SLAM-associated protein (SAP)). Mutations in SH2D1A are found in X-linked lymphoproliferative syndrome and non-Hodgkin's lymphomas. Here we report that SH2D1A is expressed in tonsillar B cells and in some B lymphoblastoid cell lines, where CD150 coprecipitates with SH2D1A and SHIP. However, in SH2D1A-negative B cell lines, including B cell lines from X-linked lymphoproliferative syndrome patients, CD150 associates only with SHP-2. SH2D1A protein levels are up-regulated by CD40 cross-linking and down-regulated by B cell receptor ligation. Using GST-fusion proteins with single replacements of tyrosine at Y269F, Y281F, Y307F, or Y327F in the CD150 cytoplasmic tail, we found that the same phosphorylated Y281 and Y327 are essential for both SHP-2 and SHIP binding. The presence of SH2D1A facilitates binding of SHIP to CD150. Apparently, SH2D1A may function as a regulator of alternative interactions of CD150 with SHP-2 or SHIP via a novel TxYxxV/I motif (immunoreceptor tyrosine-based switch motif (ITSM)). Multiple sequence alignments revealed the presence of this TxYxxV/I motif not only in CD2 subfamily members but also in the cytoplasmic domains of the members of the SHP-2 substrate 1, sialic acid-binding Ig-like lectin, carcinoembryonic Ag, and leukocyte-inhibitory receptor families.     

Effects of SH1 and SH2 modifications on myosin: similarities and differences.

The properties of myosin modified at the SH2 group (Cys-697) were studied and compared with the previously reported properties of myosin modified at the SH1 group (Cys-707). 4-[N-[(iodoacetoxy)ethyl]-N methylamino]-7-nitrobenz-2-oxa-1, 3-diazole (IANBD) was used for selective modification of the SH2 group on myosin. SH2-labeled heavy meromyosin (SH2-HMM), similar to SH1-labeled HMM (SH1-HMM), did not propel actin filaments in the in vitro motility assays. SH1- and SH2-HMM produced similar amounts of load in the mixtures with unmodified HMM; the sliding speed of actin filaments gradually decreased with an increase in the fraction of either one of the modified HMMs in the mixture. In analogy to SH1-labeled myosin subfragment 1 (SH1-S1), SH2-labeled S1 (SH2-S1) activated regulated actin in the in vitro motility assays. SH2 modification inhibited Mg-ATPase of S1 and its activation by actin. The weak binding of S1 to actin was unaffected whereas the strong binding was weakened by SH2 modification. Overall, our results demonstrate similar behavior of SH1- and SH2-modified myosin heads in the in vitro motility assays despite some differences in their enzymatic properties. The effects of these modifications are ascribed to the location of the SH1-SH2 helix relative to other functional centers of S1.     

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple‐mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two‐orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross‐reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr‐containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.     

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.