Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells.

STAT5A is a molecular regulator of proliferation, differentiation, and apoptosis in lymphohematopoietic cells. Here we show that STAT5A can serve as a functional substrate of Bruton's tyrosine kinase (BTK). Purified recombinant BTK was capable of directly binding purified recombinant STAT5A with high affinity (K(d) = 44 nm), as determined by surface plasmon resonance using a BIAcore biosensor system. BTK was also capable of tyrosine-phosphorylating ectopically expressed recombinant STAT5A on Tyr(694) both in vitro and in vivo in a Janus kinase 3-independent fashion. BTK phosphorylated the Y665F, Y668F, and Y682F,Y683F mutants but not the Y694F mutant of STAT5A. STAT5A mutations in the Src homology 2 (SH2) and SH3 domains did not alter the BTK-mediated tyrosine phosphorylation. Recombinant BTK proteins with mutant pleckstrin homology, SH2, or SH3 domains were capable of phosphorylating STAT5A, whereas recombinant BTK proteins with SH1/kinase domain mutations were not. In pull-down experiments, only full-length BTK and its SH1/kinase domain (but not the pleckstrin homology, SH2, or SH3 domains) were capable of binding STAT5A. Ectopically expressed BTK kinase domain was capable of tyrosine-phosphorylating STAT5A both in vitro and in vivo. BTK-mediated tyrosine phosphorylation of ectopically expressed wild type (but not Tyr(694) mutant) STAT5A enhanced its DNA binding activity. In BTK-competent chicken B cells, anti-IgM-stimulated tyrosine phosphorylation of STAT5 protein was prevented by pretreatment with the BTK inhibitor LFM-A13 but not by pretreatment with the JAK3 inhibitor HI-P131. B cell antigen receptor ligation resulted in enhanced tyrosine phosphorylation of STAT5 in BTK-deficient chicken B cells reconstituted with wild type human BTK but not in BTK-deficient chicken B cells reconstituted with kinase-inactive mutant BTK. Similarly, anti-IgM stimulation resulted in enhanced tyrosine phosphorylation of STAT5A in BTK-competent B cells from wild type mice but not in BTK-deficient B cells from XID mice. In contrast to B cells from XID mice, B cells from JAK3 knockout mice showed a normal STAT5A phosphorylation response to anti-IgM stimulation. These findings provide unprecedented experimental evidence that BTK plays a nonredundant and pivotal role in B cell antigen receptor-mediated STAT5A activation in B cells.     

Direct interaction of STAT4 with the IL-12 receptor.

Signal transduction by interleukin-12 (IL-12) requires phosphorylation and activation of STAT4. Direct interaction of the SH2 domain of STAT4 with a phosphotyrosine residue in the IL-12 receptor has been proposed to be required for the subsequent STAT4 phosphorylation. The IL-12 receptor beta2 subunit contains three tyrosine residues in its cytoplasmic domain. To test the hypothesis that one of these tyrosines is involved in binding STAT4, phosphopeptides were synthesized according to the amino acid sequences surrounding each of these tyrosine residues. Only the phosphopeptide containing pTyr800 strongly bound to STAT4 in a cell-free binding assay. When this phosphopeptide was introduced into TALL-104 cells, it blocked IL-12-induced STAT4 phosphorylation by competing with the IL-12 receptor for binding to STAT4. A series of alanine replacements was performed in this phosphopeptide to elucidate which amino acids surrounding the pTyr800 residue are critical for STAT4 binding. To summarize, the site on the IL-12 receptor which binds STAT4 can be described as -T-X-X-G-pY(800)-L-, where the core G-pY(800)-L motif is critical for the binding; the threonine at the pY-4 position has only a minor contribution and X represents amino acids not critical for the binding. These results demonstrate that only a small region of the IL-12 receptor is critically involved in binding STAT4 and suggest the feasibility that small molecule inhibitors could be identified which interfere with IL-12 signal transduction for treatment of autoimmune diseases.     

Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes.

pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states. We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein p130(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from p130(CAS) can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms.     

Stat2 binding to the interferon-alpha receptor 2 subunit is not required for interferon-alpha signaling.

The interferon-alpha (IFNalpha) receptor consists of two subunits, the IFNalpha receptor 1 (IFNaR1) and 2 (IFNaR2) chains. Following ligand binding, IFNaR1 is phosphorylated on tyrosine 466, and this site recruits Stat2 via its SH2 domain. In contrast, IFNaR2 binds Stat2 constitutively. In this study we have characterized the Stat2-IFNaR2 interaction and examined its role in IFNalpha signaling. Stat2 binds the major IFNaR2 protein but not a variant containing a shorter cytoplasmic domain. The interaction does not require a STAT SH2 domain. Both tyrosine-phosphorylated and non-phosphorylated Stat2 bind IFNaR2 in vitro; however, relatively little phosphorylated Stat2 associates with IFNaR2 in vivo. In vitro binding assays defined IFNaR2 residues 418-444 as the minimal interaction domain and site-specific mutation of conserved acidic residues within this domain disrupted in vitro and in vivo binding. An IFNaR2 construct carrying these mutations was either (i) overexpressed in 293T cells or (ii) used to complement IFNaR2-deficient U5A cells. Unexpectedly, the activity of an IFNalpha-dependent reporter gene was not reduced but, instead, was enhanced up to 2-fold. This suggests that this particular IFNaR2-Stat2 interaction is not required for IFNalpha signaling, but might act to negatively inhibit signaling. Finally, a doubly truncated recombinant fragment of Stat2, spanning residues 136-702, associated with IFNaR2 in vitro, indicating that the interaction with IFNaR2 is direct and occurs in a central region of Stat2 marked by a hydrophobic core.     

Genome wide analysis of pathogenic SH2 domain mutations

The authors have made a genome-wide analysis of mutations in Src homology 2 (SH2) domains associated with human disease. Disease-causing mutations have been detected in the SH2 domains of cytoplasmic signaling proteins Bruton tyrosine kinase (BTK), SH2D1A, Ras GTPase activating protein (RasGAP), ZAP-70, SHP-2, STAT1, STAT5B, and the p85alpha subunit of the PIP3. Mutations in the BTK, SH2D1A, ZAP70, STAT1, and STAT5B genes have been shown to cause diverse immunodeficiencies, whereas the mutations in RASA1 and PIK3R1 genes lead to basal carcinoma and diabetes, respectively. PTPN11 mutations cause Noonan sydrome and different types of cancer, depending mainly on whether the mutation is inherited or sporadic. We collected and analyzed all known pathogenic mutations affecting human SH2 domains by bioinformatics methods. Among the investigated protein properties are sequence conservation and covariance, structural stability, side chain rotamers, packing effects, surface electrostatics, hydrogen bond formation, accessible surface area, salt bridges, and residue contacts. The majority of the mutations affect positions essential for phosphotyrosine ligand binding and specificity. The structural basis of the SH2 domain diseases was elucidated based on the bioinformatic analysis.     

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.     

Potent inhibitory ligands of the GRB2 SH2 domain from recombinant peptide libraries.

We cloned and expressed the SH2 domain of human GRB2 as glutathione S-transferase and maltose binding protein fusion proteins. We screened three phagemid-based fd pVIII-protein phage display libraries against SH2 domain fusion proteins. Sequence analysis of the peptide extensions yielded a variety of related peptides. By examining the ability of the phage clones to bind other SH2 domains, we demonstrated that the phage were specific for the SH2 domain of GRB2. Based on the sequence motif identified in the "random" library screening experiment, we also built and screened a phage display library based on a Tyr-X-Asn motif (X5-Tyr-X-Asn-X8). To examine the affinity of the phage derived peptides for GRB2, we set up a radioligand competition binding assay based on immobilized GRB2 and radiolabelled autophosphorylated EGFR ICD as the radioligand. Results obtained with peptide competitors derived from the phage sequences demonstrated that nonphosphotyrosine-containing peptides identified with the phage display technology had an affinity for the receptor similar to tyrosine-phosphorylated peptides derived from the EGFR natural substrate. Interestingly, when the phage display peptides were then phosphorylated on tyrosine, their affinity for GRB2 increased dramatically. We also demonstrated the ability of the peptides to block the binding of the GRB2 SH2 domain to EGFR in a mammalian cell-based binding assay.     

A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction.

SH2 (src homology region 2) domains are implicated in protein-protein interactions involved in signal transduction pathways. Isolated SH2 domains bind proteins that are tyrosine phosphorylated. A novel, phosphotyrosine-independent binding interaction between BCR, the Philadelphia chromosome breakpoint cluster region gene product, and the SH2 domain of its translocation partner c-ABL has recently been reported. We have examined the ability of additional SH2 domains to bind phosphotyrosine-free BCR and compared this with their ability to bind tyrosine-phosphorylated c-ABL 1b. Of 11 individual SH2 domains examined, 8 exhibited relatively high affinity for c-ABL 1b, whereas only 4 exhibited relatively high affinity for BCR. Binding of tyrosine-phosphorylated c-ABL 1b by the relatively high-affinity ABL and ARG SH2 domains was quantitatively analyzed, and equilibrium dissociation constants for both interactions were estimated to be in the range of 5 x 10(-7) M. The ABL SH2 domain exhibited relatively high affinity for phosphotyrosine-free BCR as well; however, this interaction appears to be about two orders of magnitude weaker than binding of tyrosine-phosphorylated c-ABL 1b. The ARG SH2 domain exhibited relatively weak affinity for BCR and was determined to bind about 10-fold less strongly than the ABL SH2 domain. The ABL and ARG SH2 domains differ by only 10 of 91 amino acids, and the substitution of ABL-specific amino acids into either the amino- or carboxy-terminal half of the ARG SH2 domain was found to increase its affinity for BCR. We discuss these results in terms of a model which has been proposed for peptide binding by class I histocompatibility glycoproteins.