Jak2 tyrosine kinase mediates angiotensin II-dependent inactivation of ERK2 via induction of mitogen-activated protein kinase phosphatase 1

Previous work has shown that inhibition of Jak2 via the pharmacological compound AG490 blocks the angiotensin II (Ang II)-dependent activation of ERK2, thereby suggesting an essential role of Jak2 in ERK activation. However, recent studies have thrown into question the specificity of AG490 and therefore the role of Jak2 in ERK activation. To address this, we reconstituted an Ang II signaling system in a Jak2-/-cell line and measured the ability of Ang II to activate ERK2 in these cells. Controls for this study were the same cells expressing Jak2 via the addition of a Jak2 expression plasmid. In the cells expressing Jak2, Ang II induced a marked increase in ERK2 activity as measured by Western blot analysis and in vitro kinase assays. ERK2 activity returned to basal levels within 30 min. However, in the cells lacking Jak2, Ang II treatment resulted in ERK2 activation that did not return to basal levels until 120 min after ligand addition. Analysis of phosphatase gene expression revealed that Ang II induced mitogen-activated protein kinase phosphatase 1 (MKP-1) expression in cells expressing Jak2 but failed to induce MKP-1 expression in cells lacking Jak2. Therefore, our results suggest that Jak2 is not required for Ang II-induced ERK2 activation. Rather Jak2 is required for Ang II-induced ERK2 inactivation via induction of MKP-1 gene expression.     

The pseudokinase domain is required for suppression of basal activity of Jak2 and Jak3 tyrosine kinases and for cytokine-inducible activation of signal transduction

Janus (Jak) tyrosine kinases contain a tyrosine kinase (JH1) domain adjacent to a catalytically inactive pseudokinase domain (JH2). The JH2 domain has been implicated in regulation of Jak activity, but its function remains poorly understood. Here, we found that the JH2 domain negatively regulates the activity of Jak2 and Jak3. Deletion of JH2 resulted in increased tyrosine phosphorylation of the Jak2- and Jak3-JH2 deletion mutants as well as of coexpressed STAT5. In cytokine receptor signaling, the deletion of the Jak2- and Jak3-JH2 domains resulted in interferon-gamma and interleukin-2-independent STAT activation, respectively. However, cytokine stimulations did not further induce the JH2 deletion mutant-mediated STAT activation. The deletion of the Jak2 JH2 domain also abolished interferon-gamma-inducible kinase activation, although it did not affect the reciprocal Jak1-Jak2 interaction in 293T cells. Chimeric constructs, where the JH2 domains were swapped between Jak2 and Jak3, retained low basal activity and cytokine inducible signaling, indicating functional conservation between the two JH2 domains. However, the basal activity of Jak2 was significantly lower than that of Jak3, suggesting differences in the regulation of Jak2 and Jak3 activity. In conclusion, we found that the JH2 domain has a conserved function in Jak2 and Jak3. The JH2 domain is required for two distinct functions in cytokine signaling: (i) inhibition of the basal activity of Jak2 and Jak3, and (ii) cytokine-inducible activation of signaling. The Jak-JH2 deletion mutants are catalytically active, activate STAT5, and interact with another Jak kinase, but the JH2 domain is required to connect these signaling events to receptor activation. Thus, we propose that the JH2 domain contributes to both the uninduced and ligand-induced Jak-receptor complex, where it acts as a cytokine-inducible switch to regulate signal transduction.     

Fyn is required for haloperidol-induced catalepsy in mice

Fyn-mediated tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D(2) receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D(2) receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D(2) receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.     

The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus

The thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste) is usually produced extracellularly in Bacillus subtilis, where it is expressed as preproenzyme and subsequently processed in an autocatalytic, intramolecular process. To create the basis for the production of inactive mutants of TLP-ste, which cannot be processed in B. subtilis, we studied the expression of TLP-ste and its propeptide in cis and in trans in Escherichia coli. In contrast to thermolysin, subtilisin and alpha-lytic protease, which could be obtained only in the presence of the corresponding propeptides, TLP-ste could be produced as an active mature enzyme in E. coli in the absence of its prosequence. Surprisingly, however, a much more effective access to active mature protease was found when TLP-ste (devoid of its prosequence) was expressed as protein with an N-terminal His6 tag which accumulated in the form of inclusion bodies. Completely unexpected, the protein could be renatured from the inclusion bodies after solubilization in guanidine hydrochloride solutions in high yields. Purification to homogeneity was possible by affinity chromatography on Bacitracin silica as well as by immobilized metal ion affinity chromatography. By addition of separately expressed propeptide to the renaturation mixture yields of renaturation could not be increased significantly, confirming that the propeptide is not essential for proper folding of the enzyme or its stabilization during the folding process. Also in vivo, the expression levels of active mature TLP-ste in Escherichia coli did not significantly differ when the mature sequence was expressed alone or coexpressed with the prosequence in cis or in trans.     

A single binding motif is required for SPAK activation of the Na-K-2Cl cotransporter.

BACKGROUND: SPAK (Ste20p-related proline alanine-rich kinase) phosphorylates and activates NKCC1 (Na-K-2Cl cotransporter) in the presence of another serine/threonine kinase WNK4 (With No lysine (K)). However, whether or not the docking of SPAK to NKCC1 is a requirement for cotransporter activation has not been fully resolved. METHODS: We mutated both SPAK binding motifs in the amino-terminal tail of NKCC1 and tested the interaction between SPAK and NKCC1 using a semi in vivo yeast two-hybrid assay, (32)P-ATP in vitro phosphorylation assays, and (86)Rb(+) uptake (a K(+) congener) assays in heterologously expressed Xenopus laevis oocytes. We also used site-directed mutagenesis to identify the principle phospho-regulatory threonine residues in the amino-terminal tail of NKCC1. RESULTS: A single SPAK binding motif is necessary for isotonic NKCC1 activation. Mutation of the phenylalanine (F) residue within the motif abrogates binding and function. Phosphorylation of the cotransporter is markedly reduced in the absence of SPAK docking to NKCC1. Truncations of internal regions of the amino-terminus of NKCC1 do not disrupt protein structure enough to affect cotransporter function. Threonine residues (T(206) and T(211)) are both identified as phospho-regulatory sites of NKCC1 function. CONCLUSION: We demonstrate that physical docking of SPAK to NKCC1 is necessary for cotransporter activity under both baseline and hyperosmotic conditions. We identify T(206) and T(211) as major phospho-acceptor sites involved in cotransporter function, with T(206) common to two separate regulatory pathways: one involving SPAK, the other involving a still unknown kinase that is responsive to forskolin/PKA stimulation.     

The Drosophila Jak kinase hopscotch is required for multiple developmental processes in the eye.

Jak kinases are critical signaling components in hematopoiesis. While a large number of studies have been conducted on the roles of Jak kinases in the hematopoietic cells, much less is known about the requirements for these tyrosine kinases in other tissues. We have used loss of function mutations in the Drosophila Jak kinase Hopscotch (Hop) to determine the role of Hop in eye development. We find that Hop is required for cell proliferation/survival in the eye imaginal disc, for the differentiation of photoreceptor cells, and for the establishment of the equator and of ommatidial polarity. These results indicate that hop activity is required for multiple developmental processes in the eye, both cell-autonomously and nonautonomously.     

Interfacial catalysis by phospholipase A2: monomeric enzyme is fully catalytically active at the bilayer interface.

Interfacial catalysis in the scooting mode by phospholipase A2 (PLA2) from pancreas and venoms (18 different preparations) was examined on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol under the conditions where the rates of transbilayer and intervesicle exchanges of the enzyme, substrate, and the products of hydrolysis were negligible on the time scale (less than 30 min) on which all the substrate molecules on the outer monolayer of the target vesicles were hydrolyzed. The reaction progress curves for all PLA2s exhibited no latency period (less than 3 s). When the vesicle to PLA2 ratio in the reaction mixture was high so that according to the Poissonian distribution model at most one enzyme was bound to a vesicle, the extent of hydrolysis increased linearly with the amount of enzyme in the reaction mixture. However, the extent of hydrolysis per enzyme, NS, remained the same for all PLA2s, and it corresponded to the size of the target vesicles determined by independent methods. Similarly, the initial rate of hydrolysis increased linearly with the enzyme concentration, and the slope of the log-log plot was one under the conditions of one or more enzyme per vesicle. Such observations showed that monomeric PLA2 is fully catalytically active at the interface. This conclusion was supported by the absence of intermolecular resonance energy transfer from tryptophan-3 donor in the native PLA2 to the anthraniloyl acceptor in An87-PLA2, the catalytically active derivative of PLA2 with an anthraniloyl fluorophore on lysine 87. In this system, intermolecular resonance energy transfer was seen only when the donor-acceptor molecules were "crowded" at a high surface density with a relatively low lipid to protein mole ratio. On the basis of these results, it was concluded that secretory PLA2s from venoms and pancreas are fully catalytically active as monomers. Additional studies reported here showed that acylation of PLA2 was not necessary for catalysis or binding to the interface and that the binding of the substrate to the active site of PLA2 was not necessary for the binding of the enzyme to the interface.     

TIMP-2 is required for efficient activation of proMMP-2 in vivo

Matrix metalloproteinases (MMPs) are synthesized as latent proenzymes. A proteolytic cleavage event involving processing of the cysteine-rich N-terminal propeptide is required for their full activation. Previous in vitro studies indicated that activation of proMMP-2 can occur through formation of a trimolecular complex between MMP-14, TIMP-2, and proMMP-2 at the cell surface. Using TIMP-2-deficient mice and cells derived from them, TIMP-2 was shown to be required for efficient proMMP-2 activation both in vivo and in vitro. The requirement for TIMP-2 was not cell-autonomous as exogenously added TIMP-2 could restore activation of proMMP-2 to TIMP-2-deficient cells. Mutant mice were overtly normal, viable, and fertile on the C57BL/6 background, indicating that both TIMP-2 and activated proMMP-2 are dispensable for normal development.     

The protein-tyrosine phosphatase SHP-2 is required during angiotensin II-mediated activation of cyclin D1 promoter in CHO-AT1A cells

Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary (CHO) cells stably expressing a rat vascular angiotensin II type 1A receptor (CHO-AT(1A)). Cyclin D1 protein expression is regulated by mitogens, and its assembly with the cyclin-dependent kinases induces phosphorylation of the retinoblastoma protein pRb, a critical step in G(1) to S phase cell cycle progression contributing to the proliferative responses. In the present study, we found that in CHO-AT(1A) cells, Ang II induced a rapid and reversible tyrosine phosphorylation of various intracellular proteins including the protein-tyrosine phosphatase SHP-2. Ang II also induced cyclin D1 protein expression in a phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner. Using a pharmacological and a co-transfection approach, we found that p21(ras), Raf-1, phosphatidylinositol 3-kinase and also the catalytic activity of SHP-2 and its Src homology 2 domains are required for cyclin D1 promoter/reporter gene activation by Ang II through the regulation of MAPK/ERK activity. Our findings suggest for the first time that SHP-2 could play an important role in the regulation of a gene involved in the control of cell cycle progression resulting from stimulation of a G protein-coupled receptor independently of epidermal growth factor receptor transactivation.     

Protein truncation is required for the activation of the c-myb proto-oncogene.

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.     

A viral phospholipase A2 is required for parvovirus infectivity.

Sequence analysis revealed phospholipase A2 (PLA2) motifs in capsid proteins of parvoviruses. Although PLA2 activity is not known to exist in viruses, putative PLA2s from divergent parvoviruses, human B19, porcine parvovirus, and insect GmDNV (densovirus from Galleria mellonella), can emulate catalytic properties of secreted PLA2. Mutations of critical amino acids strongly reduce both PLA2 activity and, proportionally, viral infectivity, but cell surface attachment, entry, and endocytosis by PLA2-deficient virions are not affected. PLA2 activity is critical for efficient transfer of the viral genome from late endosomes/lysosomes to the nucleus to initiate replication. These findings offer the prospect of developing PLA2 inhibitors as a new class of antiviral drugs against parvovirus infections and associated diseases.     

Tel2 is required for activation of the Mrc1-mediated replication checkpoint

Proteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2Delta strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1Delta cells especially at S phase. In contrast, the cds1Deltaswi1Delta mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity.