A structure-function perspective of Jak2 mutations and implications for alternate drug design strategies: the road not taken.

Jak2 is a non-receptor tyrosine kinase that is involved in the control of cellular growth and proliferation. Due to its significant role in hematopoiesis, Jak2 is a frequent target for mutations in cancer, especially myeloid leukemia, lymphoid leukemia and the myeloproliferative neoplasms (MPN). These mutations are common amongst different populations all over the world and there is a great deal of effort to develop therapeutic drugs for the affected patients. Jak2 mutations, whether they are point, deletion, or gene fusion, most commonly result in constitutive kinase activation. Here, we explore the structure-function relation of various Jak2 mutations identified in cancer and understand how they disrupt Jak2 regulation. Current Jak2 inhibitors target the highly conserved active site in the kinase domain and therefore, these inhibitors may lack specificity. Based on our knowledge regarding structure-function correlations as they pertain to regulation of Jak2 kinase activity, an alternative approach for specific Jak2 targeting could be via allosteric inhibitor design. Successful reports of allosteric inhibitors developed against other kinases provide precedent for the development of Jak2 allosteric inhibitors. Here, we suggest plausible target sites in the Jak2 structure for allosteric inhibition. Such targets include the type II inhibitor pocket and substrate binding site in the kinase domain, the kinase-pseudokinase domain interface, SH2-JH2 linker region and the FERM domain. Thus, future Jak2 inhibitors that target these sites via allosteric mechanisms may provide alternative therapeutic strategies to existing ATP competitive inhibitors.

The recent medicinal chemistry development of Jak2 tyrosine kinase small molecule inhibitors.

Since the discovery of the Jak2-V617F mutation as the causative agent in a large number of myeloproliferative neoplasms (MPNs), there has been a drive to develop Jak2 specific inhibitors that can be used in therapy for MPN patients and other Jak2-related pathologies. Over the past few years, a number of research groups have sought to develop Jak2 tyrosine kinase inhibitors. These compounds are currently in pre-clinical or clinical trials. Unfortunately, there is still a need for more potent, specific, and orally bioavailable drugs to treat these diseases. Within the past twelve months, a variety of medicinal chemistry techniques have produced several lead compounds that exhibit promising Jak2 inhibitory properties. The majority of these inhibitors target the Jak2 kinase domain in general and the ATP-binding pocket in particular. In this review, we summarize these studies and discuss the structure activity relationship (SAR) properties of several compounds. As we learn more about the key structural components that provide potency and specificity in Jak2 inhibition, we will come closer to finding suitable treatment options for individuals suffering from Jak2-mediated pathologies.

The angiotensin II-dependent nuclear translocation of Stat1 is mediated by the Jak2 protein motif 231YRFRR.

In response to angiotensin II, Jak2 autophosphorylates and binds the angiotensin II AT(1) receptor. By studying a variety of Jak2 deletion proteins, we now show that the Jak2 protein motif (231)YRFRR is required for the co-association of this kinase with the AT(1) receptor. We also used a full-length Jak2 protein containing a (231)FAAAA amino acid substitution. Although this protein still autophosphorylated in response to angiotensin II, it did not co-associate with the AT(1) receptor. This uncoupling indicates that AT(1)/Jak2 co-association is not necessary for angiotensin II-induced Jak2 autophosphorylation and that Jak2 autophosphorylation per se is insufficient for AT(1) receptor co-association. In response to angiotensin II, the Jak2-(231)FAAAA mutant will tyrosine phosphorylate Stat1. However, in the absence of AT(1)/Jak2 co-association, Stat1 did not translocate into the cell nucleus and failed to mediate gene transcription. This notable result indicates that Stat1 tyrosine phosphorylation alone is insufficient for Stat1 nuclear translocation. In summary, we now show that, although Jak2-mediated tyrosine phosphorylation of Stat1 is independent of receptor co-association, Jak2-mediated recruitment of Stat1 to the AT(1) receptor is critical for Stat1 nuclear translocation and subsequent gene transcription.

The role of Ca2+ mobilization and heterotrimeric G protein activation in mediating tyrosine phosphorylation signaling patterns in vascular smooth muscle cells.

This work investigated the role of Ca2+ mobilization and heterotrimeric G protein activation in mediating angiotensin II-dependent tyrosine phosphorylation signaling patterns. We demonstrate that the predominant, angiotensin II-dependent, tyrosine phosphorylation signaling patterns seen in vascular smooth muscle cells are blocked by the intracellular Ca2+ chelator BAPTA-AM, but not by the Ca2+ channel blocker verapamil. Activation of heterotrimeric G proteins with NaF resulted in a divergent signaling effect; NaF treatment was sufficient to increase tyrosine phosphorylation levels of some proteins independent of angiotensin II treatment. In the same cells, NaF alone had no effect on other cellular proteins, but greatly potentiated the ability of angiotensin II to increase the tyrosine phosphorylation levels of these proteins. Two proteins identified in these studies were paxillin and Jak2. We found that NaF treatment alone, independent of angiotensin II stimulation, was sufficient to increase the tyrosine phosphorylation levels of paxillin. Furthermore, the ability of either NaF and/or angiotensin II to increase tyrosine phosphorylation levels of paxillin is critically dependent on intracellular Ca2+. In contrast, angiotensin II-mediated Jak2 tyrosine phosphorylation was independent of intracellular Ca2+ mobilization and extracellular Ca2+ entry. Thus, our data suggest that angiotensin II-dependent tyrosine phosphorylation signaling cascades are mediated through a diverse set of signaling pathways that are partially dependent on Ca2+ mobilization and heterotrimeric G protein activation.

Jak2 acts as both a STAT1 kinase and as a molecular bridge linking STAT1 to the angiotensin II AT1 receptor.

Angiotensin II activates the Jak-STAT pathway via the AT(1) receptor. We studied two mutant AT(1) receptors, termed M5 and M6, that contain Y to F substitutions for the tyrosine residues naturally found in the third intracellular loop and the carboxyl terminus. After binding ligand, both the M5 and M6 AT(1) receptors trigger STAT1 tyrosine phosphorylation equivalent to that observed with the wild type receptor, indicating that angiotensin II-mediated phosphorylation of STAT1 is independent of these receptor tyrosine residues. In response to angiotensin II, Jak2 autophosphorylates on tyrosine, and Jak2 and STAT1 physically associate, a process that depends on the SH2 domain of STAT1 in vitro. Evaluation of the wild type, M5, and M6 AT(1) receptors showed that angiotensin II-dependent AT(1) receptor-Jak2-STAT1 complex formation is dependent on catalytically active Jak2, not on the receptor tyrosine residues in the third intracellular loop and carboxyl tail. Immunodepletion of Jak2 virtually eliminated the ligand-dependent binding of STAT1 to the AT(1) receptor. These data indicate that the association of STAT1 with the AT(1) receptor is not strictly bimolecular; it requires Jak2 as both a STAT1 kinase and as a molecular bridge linking STAT1 to the AT(1) receptor.

A catalytically active Jak2 is required for the angiotensin II-dependent activation of Fyn.

Recent work with interleukins has shown a convergence of tyrosine phosphorylation signal transduction cascades at the level of the Janus and Src families of tyrosine kinases. Here we demonstrate that activation of the seven-transmembrane AT(1) receptor by angiotensin II induces a physical association between Jak2 and Fyn, in vivo. This association requires the catalytic activity of Jak2 but not Fyn. Deletion studies indicate that the region of Jak2 that binds Fyn is located between amino acids 1 and 240. Studies of the Fyn SH2 and SH3 domains demonstrate that the SH2 domain plays the primary role in Jak2/Fyn association. Not surprisingly, this domain shows a marked preference for tyrosine-phosphorylated Jak2. Surface plasmon resonance estimated the dissociation equilibrium constant (K(d)) of this association to be 2.36 nM. Last, in vivo studies in vascular smooth muscle cells show that, in response to angiotensin II, Jak2 activation is required for Fyn activation and induction of the c-fos gene. The significance of these data is that Jak2, in addition to serving as a critical angiotensin II activated signal transduction kinase, also functions as a docking protein and participates in the activation of Fyn by providing phosphotyrosine residues that bind the SH2 domain of Fyn.

The angiotensin II-dependent association of Jak2 and c-Src requires the N-terminus of Jak2 and the SH2 domain of c-Src.

The binding of angiotensin II (Ang II) to AT1 is known to increase the kinase activity of several nonreceptor tyrosine kinases including Jak2 and c-Src. In the present study, we demonstrate that treatment of vascular smooth muscle cells with Ang II results in a rapid and transient association of Jak2 and c-Src. This association is dependent on a catalytically active Jak2 kinase, because it is blocked both by pharmacological means and by the inability of a catalytically inactive Jak2 to associate with c-Src. c-Src bound tyrosine phosphorylated Jak2 but was unable to bind an equal amount of unphosphorylated Jak2 protein, indicating that the SH2 domain of c-Src mediates this association. In vivo studies indicated that c-Src binds the N-terminus of Jak2 as expression of a Jak2 molecule lacking the initial 240 amino acids, including 16 tyrosines, and was unable to bind c-Src. Lastly, using transiently transfected COS-7 cells, we found that Ang II treatment induced an association between c-Src and wild-type Jak2 but not between c-Src and the Jak2 molecule that lacks the initial 240 amino acids. Thus, our data suggest that in addition to increasing the kinase activities Jak2 and c-Src, treatment of cells with Ang II results in the physical association of Jak2 and c-Src; an association that is mediated by the SH2 domain of c-Src and the N-terminus of Jak2.

Janus kinase 2 (Jak2) must be catalytically active to associate with the AT1 receptor in response to angiotensin II.

Angiotensin II evokes a variety of biological responses by binding to a seven transmembrane cell surface receptor termed AT1. Ligand binding to the AT1 receptor induces the physical association and activation of the intracellular kinase Jak2. To elucidate the mechanism of this association, COS-7 cells were co-transfected with the AT1 receptor and either wild type Jak2 or a catalytically inactive Jak2. AT1 receptor-Jak2 association was assessed in vitro by a GST-AT1 receptor fusion protein binding assay and in vivo by direct co-immunoprecipitation of the receptor-Jak2 complex. Both studies showed that Jak2 must be catalytically active to form a complex with the AT1 receptor, and that complex formation is associated with Jak2 tyrosine phosphorylation. These results were confirmed using the Jak2 specific inhibitor AG-490. We also found that over-expression of wild type Jak2 in COS-7 cells leads to in vivo complex formation of spontaneously autophosphorylated Jak2 with the AT1 receptor. No such complex formation was observed with a dominant negative Jak2. Thus, the physical association of Jak2 with the AT1 receptor is regulated by an angiotensin II mediated autophosphorylation event.

Phosphorylation of p130Cas by angiotensin II is dependent on c-Src, intracellular Ca2+, and protein kinase C.

p130Cas is a signaling molecule that was initially found to be tyrosine-phosphorylated in v-Crk and v-Src transformed cells. We characterized the regulation of p130Cas tyrosine phosphorylation in vascular smooth muscle cells by angiotensin II (Ang II). This ligand induced a transient increase in p130Cas tyrosine phosphorylation, which was sensitive to the actin polymerization inhibitor cytochalasin D and to the intracellular Ca2+ chelator BAPTA-AM but not the Ca2+ channel blocker verapamil. The Ang II-induced tyrosine phosphorylation of p130Cas was also dependent on an active Src family tyrosine kinase, since it could be blocked by the Src kinase inhibitors geldanamycin and PP1. Ang II treatment resulted in the ability of p130Cas to bind at least 11 different phosphate-containing proteins. Analysis of these proteins revealed that protein kinase Calpha and the cell adhesion signaling molecule pp120 formed temporal associations with p130Cas in response to Ang II. c-Src was found to associate with p130Cas in a manner that was independent of Ang II treatment. Inhibition of protein kinase C by either calphostin C or phorbol 12-myristate 13-acetate downregulation inhibited the Ang II-induced tyrosine phosphorylation of p130Cas. These results are the first to demonstrate that the tyrosine phosphorylation of p130Cas by Ang II is transduced by the Src, intracellular Ca2+, protein kinase C signaling pathway.

Angiotensin II signal transduction pathways.

It has been 100 years since the discovery of renin by Tigerstedt and Bergman. Since that time, numerous discoveries have advanced our understanding of the renin-angiotensin system, including the observation that angiotensin II is the effector molecule of this system. A remarkable aspect of angiotensin II is the many different physiological responses this simple peptide induces in different cell types. Here, we focus on the signal transduction pathways that are activated as a consequence of angiotensin II binding to the AT1 receptor. Classical signaling pathways such as the activation of heterotrimeric G proteins by the AT1 receptor are discussed. In addition, recent work examining the role of tyrosine phosphorylation in angiotensin II-mediated signal transduction is also examined. Understanding how these distinct signaling pathways transduce signals from the cell surface will advance our understanding of how such a simple molecule elicits such a wide variety of specific cellular responses.

Dependence on the motif YIPP for the physical association of Jak2 kinase with the intracellular carboxyl tail of the angiotensin II AT1 receptor.

Angiotensin II is the effector molecule of the renin-angiotensin system. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT1. These receptors contain the structural features of the seven-transmembrane, G-protein-coupled receptor superfamily. Angiotensin II, acting through the AT1 receptor, also stimulates the Jak/STAT pathway by inducing ligand-dependent Jak2 tyrosine phosphorylation and activation. Here, we show that a glutathione S-transferase fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT1A receptor physically binds to Jak2 in an angiotensin II-dependent manner. Deletional analysis, using both in vitro protocols and cell transfection analysis, showed that this association is dependent on the AT1A receptor motif YIPP (amino acids 319-322). The wild-type AT1A receptor can induce Jak2 tyrosine phosphorylation. In contrast, an AT1A receptor lacking the YIPP motif is unable to induce ligand-dependent phosphorylation of Jak2. Competition experiments with synthetic peptides suggest that each of the YIPP amino acids, including tyrosine 319, is important in Jak2 binding to the AT1A receptor. The binding of the AT1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors.

Cloning and partial characterization of the mouse glutamine:fructose-6-phosphate amidotransferase (GFAT) gene promoter.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the enzyme that is rate limiting in the synthesis of glucosamine and hexosamines. Glucosamine has been proposed to contribute to the glucotoxicity of diabetes. Evidence that the gene encoding GFAT is transcriptionally regulated prompted us to clone and characterize its promoter. The position of the mouse GFAT promoter relative to the translational start site was located by primer extension and found to be 149 bp upstream of the translational start site. A 1.9 kb SacI fragment of the GFAT gene was found to contain the promoter and 88 bp of sequence downstream of the transcriptional start site. This promoter segment could drive expression of a luciferase reporter gene, could confer correct transcriptional initiation to the reporter and could confer the EGF-responsiveness previously observed in the native gene. The mouse GFAT promoter lacks a canonical TATA box and has several GC boxes within a highly GC-rich region. Deletional analysis of the promoter indicated that a proximal element extending to -120 relative to the transcriptional start site could confer reporter expression at a level of 57% of the 1.9 kb construct. Detailed analysis of this proximal region by DNase I footprinting, electrophoretic mobility shift assays and site-directed mutagenesis indicated that Sp1 binds to three elements in this proximal promoter segment and plays a vital role in regulation of transcription from this gene.

Role of intracellular calcium in the angiotensin II-mediated tyrosine phosphorylation and dephosphorylation of PLC-gamma 1.

Angiotensin II induces the rapid temporal tyrosine phosphorylation and activation of phospholipase C-gamma 1 (PLC-gamma 1) and the elevation of intracellular calcium levels. To investigate the relationship of these intracellular signaling events, rat aortic smooth muscle cells were treated with the calcium chelator BAPTA-AM, the calcium channel blocker verapamil, the intracellular calcium antagonist TMB-8, and the calcium ionophore ionomycin. The effects of these agents on PLC-gamma 1 tyrosine phosphorylation were then measured. We found that treatment of these cells with the calcium inhibitors augmented the basal level of PLC-gamma 1 tyrosine phosphorylation, without changing the peak level of tyrosine phosphorylation induced by angiotensin II. The rapid dephosphorylation of PLC-gamma 1 that follows angiotensin II stimulation was prevented by these calcium antagonists. In contrast, angiotensin II-induced tyrosine phosphorylation of PLC-gamma 1 was inhibited by ionomycin. These results suggest that the angiotensin II-induced tyrosine phosphorylation of PLC-gamma 1 is calcium-independent, while the dephosphorylation is calcium-dependent.

Glucose metabolism to glucosamine is necessary for glucose stimulation of transforming growth factor-alpha gene transcription.

Transforming growth factor-alpha (TGFalpha) gene transcription can be increased when arterial smooth muscle cells are exposed to supraphysiological concentrations of glucose, and this effect of glucose can be mimicked by glucosamine. To determine whether the metabolism of glucose to glucosamine is required for this glucose effect, the rate-limiting step in glucose metabolism to glucosamine through the enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) was blocked using pharmacological and antisense strategies. We found that blockage of GFAT activity or expression significantly blunted the glucose-induced increase of TGFalpha expression. Blockage of GFAT also resulted in a decreased RL2 signal on intracellular proteins as detected by Western blotting and indirect immunofluorescence. The RL2 monoclonal antibody recognizes an epitope on proteins that contain N-acetylglucosamine and thus is a measure of protein glycosylation. Conversely, treatment of the cells with glucose and glucosamine resulted in an increase in the RL2 epitope on intracellular proteins. These results indicate that the metabolism of glucose to glucosamine is necessary for the transcriptional stimulation of TGFalpha expression in vascular smooth muscle cells by glucose. Furthermore, the level of glycosylation of some intracellular proteins can be modulated in response to physiological changes in the extracellular glucose concentration and the net activity of GFAT.

The murine glutamine:fructose-6-phosphate amidotransferase-encoding cDNA sequence.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme in hexosamine synthesis and has been implicated in the control of growth factor gene expression. We cloned a mouse cDNA which is 91% homologous to the human sequence. The deduced amino-acid sequence shows 98.6% identity to human GFAT. The cDNA is derived from a 7-kb mRNA in the mouse, while there are multiple-sized human mRNAs.

Human immunodeficiency virus 1 Tat stimulates transcription of the transforming growth factor alpha gene in an epidermal growth factor-dependent manner.

The human immunodeficiency virus 1 (HIV-1) tat gene encodes a protein of critical importance for viral transcription. In addition, Tat has been shown capable of entering cells, stimulating cell proliferation, and altering host cell gene expression. We examined the effect of Tat on the expression of the transforming growth factor alpha (TGF-alpha) gene in MDA468 human breast carcinoma cells. We showed that these cells were capable of supporting the activation of the HIV-1 long terminal repeat by Tat. Then, in cotransfection assays, in which the TGF-alpha promoter was linked to a luciferase reporter gene and the tat gene was expressed under the control of the SV40 early promoter, we showed that tat gene expression increased TGF-alpha-luciferase reporter function but only in cells stimulated with epidermal growth factor (EGF). The effects of tat and EGF were dose dependent. To confirm these cotransfection data, Tat was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) and purified on glutathione-agarose. GST-Tat was introduced into the MDA468 cells either in the presence of chloroquine or by scrape loading. The biological activity of GST-Tat was tested on cells that had been stablely transfected with the HIV-1 long terminal repeat linked to luciferase as a reporter. GST-Tat was then introduced into the cells, and the level of TGF-alpha mRNA was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Triazolam fails to induce sleep in suprachiasmatic nucleus-lesioned rats.

Rats with suprachiasmatic nuclei (SCN) lesions did not show increased sleep after triazolam (TRZ) injections at any dose from 0.2 to 1.6 mg/kg, whereas 0.4 mg/kg TRZ given intact rats in the middle of their activity phase significantly increased sleep. Across SCN-lesioned and intact rats, the amount of sleep before and after TRZ 0.4 mg/kg was negatively correlated. SCN-lesioned rats did not have a circadian activity-dominant period and so did not accumulate a biological sleep debt. Their lack of response to TRZ may have resulted from the absence of a sleep debt compared to intact rats injected in the middle of their activity phase. These data support our hypothesis that the homeostatic process controlling sleep gates benzodiazepine hypnotic efficacy.