Specific dephosphorylation of Janus Kinase 2 by protein tyrosine phosphatases

Many protein kinases are activated through phosphorylation of an activation loop thereby turning on downstream signaling pathways. Activation of JAK2, a nonreceptor tyrosine kinase with an important role in growth factor and cytokine signaling, requires phosphorylation of the 1007 and 1008 tyrosyl residues. Dephosphorylation of these two sites by phosphatases presumably inactivates the enzyme, but the underlying mechanism is not known. In this study, we employed MALDI‐TOF/TOF and triple quadrupole mass spectrometers to analyze qualitatively and quantitatively the dephosphorylation process by using synthetic peptides derived from the tandem autophosphorylation sites (Y1007 and Y1008) of human JAK2. We found that tyrosine phosphatases catalyzed the dephosphorylation reaction sequentially, but different enzymes exhibited different selectivity. Protein tyrosine phosphatase 1B caused rapid dephosphorylation of Y1008 followed by Y1007, while SHP1 and SHP2 selectively dephosphorylated Y1008 only, and yet HePTP randomly removed a single phosphate from either Y1007 or Y1008, leaving behind mono‐phosphorylated peptides. The specificity of dephosphorylation was further confirmed by molecular modeling. The data reveal multiple modes of JAK2 regulation by tyrosine phosphatases, reflecting a complex, and intricate interplay between protein phosphorylation and dephosphorylation.     

Mutational analysis of stress-responsive peanut dual specificity protein kinase. Identification of tyrosine residues involved in regulation of protein kinase activity

We recently reported that Arachis hypogaea serine/threonine/tyrosine (STY) protein kinase is developmentally regulated and is induced by abiotic stresses (Rudrabhatla, P., and Rajasekharan, R. (2002) Plant Physiol. 130, 380-390). Other than MAPKs, the site of tyrosine phosphorylation has not been documented for any plant kinases. To study the role of tyrosines in the phosphorylation of STY protein kinase, four conserved tyrosine residues were sequentially substituted with phenylalanine and expressed as histidine fusion proteins. Mass spectrometry experiments showed that STY protein kinase autophosphorylated within the predicted kinase ATP-binding motif, activation loop, and an additional site in the C terminus. The protein kinase activity was abolished by substitution of Tyr(297) with Phe in the activation loop between subdomains VII and VIII. In addition, replacing Tyr(148) in the ATP-binding motif and Tyr(317) in the C-terminal domain with Phe not only obliterated the ability of the STY protein kinase protein to be phosphorylated, but also inhibited histone phosphorylation, suggesting that STY protein kinase is phosphorylated at multiple sites. Replacing Tyr(213) in the Thr-Glu-Tyr sequence motif with Phe resulted in a 4-fold increase in autophosphorylation and 2.8-fold increase in substrate phosphorylation activities. Mutants Y148F, Y297F, and Y317F displayed dramatically lower phosphorylation efficiency (k(cat)/K(m)) with ATP and histone, whereas mutant Y213F showed increased phosphorylation. Our results suggest that autophosphorylation of Tyr(148), Tyr(213), Tyr(297), and Tyr(317) is important for the regulation of STY protein kinase activity. Our study reveals the first example of Thr-Glu-Tyr domain-mediated autoinhibition of kinases.     

The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop

The Janus family of protein tyrosine kinases (JAKs) regulate cellular processes involved in cell growth, differentiation and transformation through their association with cytokine receptors. However, compared with other kinases, little is known about cellular regulators of the JAKs. We have recently identified a JAK-binding protein (JAB) that inhibits JAK signaling in cells. In the studies presented here we demonstrate that JAB specifically binds to the tyrosine residue (Y1007) in the activation loop of JAK2, whose phosphorylation is required for activation of kinase activity. Binding to the phosphorylated activation loop requires the JAB SH2 domain and an additional N-terminal 12 amino acids (extended SH2 subdomain) containing two residues (Ile68 and Leu75) that are conserved in JAB-related proteins. An additional N-terminal 12-amino-acid region (kinase inhibitory region) of JAB also contributes to high-affinity binding to the JAK2 tyrosine kinase domain and is required for inhibition of JAK2 signaling and kinase activity. Our studies define a novel type of regulation of tyrosine kinases and might provide a basis for the design of specific tyrosine kinase inhibitors.     

Simultaneous activation of JAK1 and JAK2 confers IL-3 independent growth on Ba/F3 pro-B cells

JAK1 and JAK2 are tyrosine kinases involved in the regulation of cell proliferation, differentiation, and survival. These proteins may play a key role in mediating the effects of the cytokine IL-3 on hematopoietic cells. IL-3 induces tyrosine phosphorylation of both JAK1 and JAK2. However, it is not clear whether the activation of JAK1, JAK2, or both is sufficient to confer factor-independent growth in IL-3 dependent cells. To address this issue, fusion proteins CD16/CD7/JAK (CDJAK), comprised of a CD16 extracellular domain, a CD7 transmembrane domain, and a JAK cytoplasmic region (either a wild-type JAK or a dominant negative mutant of JAK) were constructed. We established several Ba/F3 derivatives that stably overexpress the conditionally active forms of either CDJAK1, CDJAK2, or both these fusion proteins. In this study, the autophosphorylation of CDJAK1 or CDJAK2 was induced by crosslinking with anti-CD16 antibody. We demonstrated that, like their wild-type counterparts, CDJAK1 and CDJAK2 were preassociated with the IL-3 receptor beta and alpha subunits, respectively. Furthermore, the simultaneous activation of both CDJAK1 and CDJAK2 fusion proteins, but not either one alone, led to the tyrosine phosphorylation of the IL-3 receptor beta subunit, the activation of downstream signaling molecules, including STAT5, Akt, and MAPK, and the conferring of factor-independent growth to IL-3-dependent Ba/F3 cells. Coexpression of dominant negative mutants CDJAK1KE or CDJAK2KE with wild type CDJAK2 or CDJAK1, respectively, inhibited these activation activities. These results suggest that JAK1 and JAK2 must work cooperatively and not independently and that their actions are dependent on having normal kinase activity to trigger downstream signals leading to IL-3 independent proliferation and survival of Ba/F3 cells.     

Modulation of Janus kinase 2 by p53 in ovarian cancer cells

The constitutive activation of the Janus kinase 2 (JAK2) and mutation of the p53 tumor suppressor are both detected in human cancer. We examined the potential regulation of JAK2 phosphorylation by wild-type (wt) p53 in human ovarian cancer cell lines, Caov-3 and MDAH2774, which harbor mutant form of p53 tumor suppressor gene and high levels of phosphorylated JAK2. The wt p53 gene was re-introduced into the cells using an adenovirus vector. In addition to wt p53, mutant p53 22/23, mutant p53-175, and NCV (negative control virus) were introduced into the cells in the control groups. Expression of wt p53, but not that of p53-175 mutant, diminished JAK2 tyrosine phosphorylation in MDAH2774 and Caov-3 cell lines. Expression of wt p53 or p53 22/23 mutant did not cause a reduction in the phosphorylation of unrelated protein kinases, ERK1 and ERK2 (ERK1/2). The inhibition of JAK2 tyrosine phosphorylation can be reversed by tyrosine phosphatase inhibitor, sodium orthovanadate. Protein tyrosine phosphatase 1-B levels increased with introduction of wt p53 and may be involved in the dephosphorylation of JAK2. These findings present a possible p53-dependent cellular process of modulating JAK2 tyrosine phosphorylation in ovarian cancer cell lines.     

Phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase. Temporal correlation of acetylcholine's effect on site phosphorylation, enzyme activation, and catecholamine synthesis

Tryptic peptide fragments of tyrosine hydroxylase isolated from 32PO4-prelabeled bovine adrenal chromaffin cells are resolved into seven phosphopeptides by reverse phase-high performance liquid chromatography. All seven of the peptides are phosphorylated on serine residues. Three of these putative phosphorylation sites, peptides 3, 5, and 6, are rapidly phosphorylated (5-fold in 15 s) by both acetylcholine stimulation and potassium depolarization of the cells, and this phosphorylation is accompanied by a similarly rapid activation of the enzyme. Both phosphorylation and activation are transient and do not account for the prolonged increase in catecholamine biosynthesis produced by these stimuli. Peptides 4 and 7 show a much slower and sustained increase in phosphorylation (3-fold in 4 min) in response to acetylcholine and potassium. Phosphorylation of these peptides correlates with the sustained increase in catecholamine biosynthesis rather than enzyme activation. Peptides 1 and 2 are not stimulated by any agonist yet employed and thus show no relation to enzyme activation or catecholamine biosynthesis. Phosphorylation of all five peptides by acetylcholine or potassium is calcium-dependent. In contrast to the stimulation of phosphorylation of tyrosine hydroxylase on multiple sites, forskolin stimulates the phosphorylation of only peptide 6, and this is accompanied by a coordinated activation of tyrosine hydroxylase and increased catecholamine biosynthesis. These findings show that the phosphorylation of tyrosine hydroxylase in intact cells is more complex than predicted from in vitro results, that at least two protein kinases are involved in the secretagogue-induced phosphorylation of tyrosine hydroxylase, and that the regulation of catecholamine biosynthesis, in response to phosphorylation, appears to involve both tyrosine hydroxylase activation and other mechanisms.     

Expression,purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69 mg per 20 L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3 mg per 10 L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K_m and k_cat) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4 Å. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.     

Mechanistic effects of autophosphorylation on receptor tyrosine kinase catalysis: enzymatic characterization of Tie2 and phospho-Tie2

Activation of receptor tyrosine kinases by autophosphorylation is one of the most common and critical transformations in signal transduction, yet its role in catalysis remains controversial. Autophosphorylation of the angiogenic receptor tyrosine kinase Tie2 was studied in terms of the autophosphorylation sites, sequence of phosphorylation at these sites, kinetic effects, and mechanistic consequences. Isoelectric focusing electrophoresis and mass spectrometric analysis of a Tie2 autophosphorylation time course showed that Tyr992 on the putative activation loop was phosphorylated first followed by Tyr1108 in the C-terminal tail (previously unidentified autophosphorylation site). Autophosphorylation of Tie2 to produce pTie2 resulted in a 100-fold increase in k(cat) and a 460-fold increase in k(cat)/K(m). Viscosity studies showed that the unphosphorylated Tie2 was partially limited by product diffusion ((k(cat))(eta) = 0.67 +/- 0.06), while product release was more rate-limiting ((k(cat))(eta) = 0.94 +/- 0.08) for autophosphorylated Tie2 (pTie2). Furthermore, autophosphorylation did not significantly affect the phosphoacceptor dissociation constants. There was a significant (k(cat))(H)/(k(cat))(D) solvent isotope effect (SIE) for unphosphorylated Tie2 (2.42 +/- 0.12) and modest SIE (1.28 +/- 0.04) for pTie2, which is consistent with the chemistry step being more rate-limiting for Tie2 as compared to pTie2. The pH-rate profiles of Tie2 and pTie2 revealed a >0.5 unit shift in the pK(a) values of catalytically relevant ionizable residues upon autophosphorylation. The shift in rate-limiting step will result in a different distribution of enzyme pools (e.g., E, E*S, E*P, etc.) which may modulate the susceptibility to inhibition. Tie2 and pTie2 were profiled with a panel of known ATP-competitive kinase inhibitors. Tie2 activation perturbs catalytic residue ionizations, shifts the rate-limiting step to almost exclusive diffusion-control, and transforms the kinase into a more perfect catalyst.     

Ligand regulates epidermal growth factor receptor kinase specificity: activation increases preference for GAB1 and SHC versus autophosphorylation sites

The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K(m) and k(cat), revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k(cat) approximately 5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of approximately 5-fold in the specificity constants (k(cat)/K(m)) for EGFR peptides, whereas approximately 15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins approximately 4-6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.     

Substrate specificity of the oncoprotein v-Fps: site-specific mutagenesis of the putative P+1 pocket

Based on the X-ray structure of the insulin receptor kinase [Hubbard, S. R. (1997) EMBO J. 16, 5572-5581], Arg-1130 in the oncoprotein v-Fps, a nonreceptor tyrosine protein kinase, is predicted to interact with the P+1 glutamate in substrate peptides. To determine whether this residue is an important recognition element in v-Fps, Arg-1130 was substituted with leucine (R1130L) and glutamic acid (R1130E). The ability of these mutants to phosphorylate the peptide EAEIYXAIE, where X is glutamic acid, alanine, or lysine, was assessed. A comparison of the rates of peptide phosphorylation under limiting substrate concentrations (i.e., k(cat)/K(m) conditions) indicates that substrate specificity is altered by the electrostatic environment of the P+1 pocket. When the pocket displays a positive charge (Arg-1130; wild type), no charge (R1130L), or a negative charge (R1130E), v-Fps prefers to phosphorylate the glutamate peptide over the lysine peptide by a 200:1, 9:1, or 1:1 margin. While k(cat)/K(m) for the glutamate peptide is 50-fold higher for wild type compared to R1130E, k(cat)/K(m) for the lysine peptide is 3-fold higher for R1130E compared to wild type, a 150-fold change in relative substrate specificity. Analysis of the individual steps in the kinetic mechanism using viscosometric techniques indicates that the wild-type enzyme binds the glutamate peptide 3-fold better than the alanine peptide and, at least, 10-fold better than the lysine peptide. For R1130L, this margin range is reduced substantially, and for R1130E, no binding preference is observed. Nonetheless, the lysine peptide binds, at least, 4-fold better to R1130E than to wild type, and the glutamate peptide binds 3-fold poorer to R1130E than to wild type. The mutants lower the phosphoryl transfer rate by 4-30-fold for the three peptides, suggesting that Arg-1130 helps to position the tyrosine for optimum catalysis. The data indicate that a single mutation in v-Fps can alter significantly the relative substrate specificity by about 2 orders of magnitude with, at least, 50% of this effect occurring through relative changes in peptide binding affinity.     

Regulation of p27Kip1 by mitogen-induced tyrosine phosphorylation

Extracellular mitogen signal transduction is initiated by ligand binding to specific receptors of target cells. This causes a cellular response that frequently triggers the activation of tyrosine kinases. Non-receptor kinases like Src and Lyn can directly phosphorylate the Cdk inhibitor protein p27^Kip1. Tyrosine phosphorylation can cause impaired Cdk-inhibitory activity and decreased stability of p27. In addition to these non-receptor tyrosine kinases, the receptor-associated tyrosine kinase Janus kinase 2 (JAK2) was recently identified to phosphorylate p27. JAK2 becomes activated through binding of various cytokines and growth factors to their corresponding receptors and can directly bind and selectively phosphorylate tyrosine residue 88 (Y88) of the Cdk inhibitor p27. This impairs Cdk inhibition by p27 and promotes its ubiquitin-dependent proteasomal degradation. Via this mechanism, JAK2 can link cytokine and growth factor initiated signal transduction to p27 regulation, whereas oncogenes like JAK2V617F or BCR-Abl can use this mechanism to inactivate the Cdk inhibitor.     

Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition

Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parameters K(m) and k(cat) both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family.     

A single substitution of the insulin receptor kinase inhibits serine autophosphorylation in vitro: evidence for an interaction between the C-terminus and the activation loop

We examined the effects of mutations of tyrosine and serine autophosphorylation sites on the dual specificity of the insulin receptor kinase (IRKD) in vitro using autophosphorylation and substrate phosphorylation and phosphopeptide mapping. For comparable studies, the recombinant kinases were overexpressed in the baculovirus system, purified, and analyzed. The phosphate incorporation into the enzymes was in the range of 3-4.5 mol/mol, and initial velocities of autophosphorylation were reduced up to 2-fold. However, the mutation Y1151F in the activation loop inhibited phosphate incorporation in the C-terminal serine residues 1275 and 1309, due to a 10-fold decrease of the initial velocity of serine autophosphorylation. Although the K(M) and V(MAX) values of this mutant were only slightly altered in substrate phosphorylation reactions using a recombinant C-terminal insulin receptor peptide (K(M): Y1151F, 9.9 +/- 0.4 microM; IRKD, 6.1 +/- 0.2 microM; V(MAX): Y1151F, 72 +/- 4 nmol min(-)(1) mg(-)(1); IRKD, 117 +/- 6 nmol min(-)(1) mg(-)(1)), diminished phosphate incorporation into serine residues of the peptide was observed. In contrast, the phosphorylation of a recombinant IRS-1 fragment, which was shown to be phosphorylated markedly on serine residues by IRKD, was not affected by any kinase mutation. These results underline that IRKD is a kinase with dual specificity. The substrate specificity toward C-terminal serine phosphorylation sites can be modified by a single amino acid substitution in the activation loop, whereas the specificity toward IRS-1 is not affected, suggesting that the C-terminus and the activation loop interact.     

Identification of phosphorylation sites within the SH3 domains of Tec family tyrosine kinases

Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation mechanism, which for Bruton's tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3 domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains, but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however, one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other through transphosphorylation.     

Enhancement of ABL kinase catalytic efficiency by a direct binding regulator is independent of other regulatory mechanisms

ABL family tyrosine kinases are tightly regulated by autoinhibition and phosphorylation mechanisms. These kinases maintain an inactive conformation through intramolecular interactions involving SH3 and SH2 domains. RIN1, a downstream effector of RAS, binds to the ABL SH3 and SH2 domains and stimulates ABL tyrosine kinase activity. RIN1 binding to the ABL2 kinase resulted in a large decrease in Km and a small increase in Vmax toward an ABL consensus substrate peptide. The enzyme efficiency (k(cat)/Km) was increased more than 5-fold by RIN1. In addition, RIN1 strongly enhanced ABL-mediated phosphorylation of CRK, PSTPIP1, and DOK1, all established ABL substrates but with unique protein structures and distinct target sequences. Importantly RIN1-mediated stimulation of ABL kinase activity was independent of activation by SRC-mediated phosphorylation. RIN1 increased the kinase activity of both ABL1 and ABL2, and this occurred in the presence or absence of ABL regulatory domains outside the SH3-SH2-tyrosine kinase domain core. We further demonstrate that a catalytic site mutation associated with broad drug resistance, ABL1T315I, remains responsive to stimulation by RIN1. These findings are consistent with an allosteric kinase activation mechanism by which RIN1 binding promotes a more accessible ABL catalytic site through relief of autoinhibition. Direct disruption of RIN1 binding may therefore be a useful strategy to suppress the activity of normal and oncogenic ABL, including inhibitor-resistant mutants that confound current therapeutic strategies. Stimulation through derepression may be applicable to many other tyrosine kinases autoinhibited by coupled SH3 and SH2 domains.     

Two closely spaced tyrosines regulate NFAT signaling in B cells via Syk association with Vav

Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2.     

Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages

The mechanisms by which lipopolysaccharide (LPS) is recognized, and how such recognition leads to innate immune responses, are poorly understood. Stimulation with LPS induces the activation of a variety of proteins, including mitogen-activated protein kinases (MAPKs) and NF-B. Activation of protein tyrosine kinases (PTKs) is also necessary for a number of biological responses to LPS. We used a murine macrophage-like cell line, RAW264.7, to demonstrate that Janus kinase (JAK)2 is tyrosine phosphorylated immediately after LPS stimulation. Anti-Toll-like receptor (TLR)4 neutralization antibody inhibits the phosphorylation of JAK2 and the c-Jun NH₂-terminal protein kinase (JNK). Both the JAK inhibitor AG490 and the kinase-deficient JAK2 protein reduce the phosphorylation of JNK and phosphatidylinositol 3-kinase (PI3K) via LPS stimulation. Pharmacological inhibition of the kinase activity of PI3K with LY-294002 decreases the phosphorylation of JNK. Finally, we show that JAK2 is involved in the production of IL-1 and IL-6. PI3K and JNK are also important for the production of IL-1. These results suggest that LPS induces tyrosine phosphorylation of JAK2 via TLR4 and that JAK2 regulates phosphorylation of JNK mainly through activation of PI3K. Phosphorylation of JAK2 via LPS stimulation is important for the production of IL-1 via the PI3K/JNK cascade. Thus JAK2 plays a pivotal role in LPS-induced signaling in macrophages. cytokine; toll-like receptor-4; c-Jun NH₂-terminal kinase