Calcium-dependent Kinase Research Articles

Targeting lymphocyte signaling pathways as a therapeutic approach to systemic lupus erythematosus

Over the past year several key pathways in systemic lupus erythematosus (SLE) lymphocyte signaling have been identified. Pathways that can be exploited for therapy are discussed in this review. Inhibition of SLE T cell activation by blocking spleen tyrosine kinase (Syk) and SLE T cell migration by blocking CD44 or CXCR4 lead to amelioration of lupus in lupus-prone mice. Similar results can be achieved by boosting CD8+ Treg numbers. Small molecules that block the kinases CaMKIV (calcium and calmodulin dependent kinase IV) and Bruton Tyrosine kinase (Btk) and the phosphatase calcineurin were shown to be effective in treating murine lupus. Finally, gene methylation status determines the expression of several key genes in SLE and strategies to correct it have shown promising results in preclinical studies. Molecules that enhance T cell receptor (TCR) signaling or increase lymphocyte migration can be inhibited successfully with significant improvement of disease intensity in lupus-prone mice using small molecules. Manipulation of promoter methylation and histone acetylation represents a novel way to alter gene transcription in SLE.

Apoptosis and Autophagy: Decoding Calcium Signals that Mediate Life or Death

Calcium is a versatile and dynamic 2nd messenger that is essential for the survival of all higher organisms. In cells that undergo activation or excitation, calcium is released from the endoplasmic/sarcoplasmic reticulum to activate calcium-dependent kinases and phosphatases, thereby regulating numerous cellular processes; for example, apoptosis and autophagy. In the case of apoptosis, endogenous ligands or pharmacological agents induce prolonged cytosolic calcium elevation, which in turn leads to cell death. In contrast, there is now evidence that calcium regulates autophagy by several mechanisms, and these may be important for maintaining cell survival. Here we summarize what is known about how calcium regulates these life and death decisions. We pay particular attention to pathways that have been described in lymphocytes and cardiomyocytes, as these systems provide optimal models for understanding calcium signaling in the context of normal cell physiology.

Essential Role of Nuclear Factor of Activated T Cells (NFAT)-mediated Wnt Signaling in Osteoblast Differentiation Induced by Strontium Ranelate

The antiosteoporotic treatment strontium ranelate (SrRan) was shown to increase bone mass and strength by dissociating bone resorption and bone formation. To identify the molecular mechanisms of action of SrRan on osteoblasts, we investigated its effects on calcineurin-NFAT (nuclear factor of activated T cells) signaling, an important calcium sensitive pathway controlling bone formation. Using murine MC3T3-E1 and primary murine osteoblasts, we demonstrate that SrRan induces NFATc1 nuclear translocation, as shown by immunocytochemical and Western blot analyses. Molecular analysis showed that SrRan increased NFATc1 transactivation in osteoblasts, an effect that was fully abrogated by the calcineurin inhibitors cyclosporin A or FK506, confirming that SrRan activates NFATc1 signaling in osteoblasts. This has functional implications because calcineurin inhibitors blunted the enhanced osteoblast replication and expression of the osteoblast phenotypic markers Runx2, alkaline phosphatase, and type I collagen induced by SrRan. We further found that SrRan increased the expression of Wnt3a and Wnt5a as well as beta-catenin transcriptional activity in osteoblasts, and these effects were abolished by calcineurin inhibitors. The Wnt inhibitors sFRP1 and DKK1 abolished SrRan-induced osteoblast gene expression. Furthermore, blunting the Wnt5a receptor Ryk or RhoA that acts downstream of Ryk abrogated cell proliferation and osteoblast gene expression induced by SrRan. These results indicate that activation of NFATc1 and downstream canonical and non-canonical Wnt signaling pathways mediate SrRan-induced osteoblastic cell replication and differentiation, which provides novel insights into the mechanisms of action of this antiosteoporotic agent in osteoblastogenesis.

GsCBRLK, a calcium/calmodulin-binding receptor-like kinase, is a positive regulator of plant tolerance to salt and ABA stress

Calcium/calmodulin-dependent kinases play vital roles in protein phosphorylation in eukaryotes, yet little is known about the phosphorylation process of calcium/calmodulin-dependent protein kinase and its role in stress signal transduction in plants. A novel plant-specific calcium-dependent calmodulin-binding receptor-like kinase (GsCBRLK) has been isolated from Glycine soja. A subcellular localization study using GFP fusion protein indicated that GsCBRLK is localized in the plasma membrane. Binding assays demonstrated that calmodulin binds to GsCBRLK with an affinity of 25.9 nM in a calcium-dependent manner and the binding motif lies between amino acids 147 to169 within subdomain II of the kinase domain. GsCBRLK undergoes autophosphorylation and Myelin Basis Protein phosphorylation in the presence of calcium. It was also found that calcium/calmodulin positively regulates GsCBRLK kinase activity through direct interaction between the calmodulin-binding domain and calmodulin. So, it is likely that GsCBRLK responds to an environmental stimulus in two ways: by increasing the protein expression level and by regulating its kinase activity through the calcium/calmodulin complex. Furthermore, cold, salinity, drought, and ABA stress induce GsCBRLK gene transcripts. Over-expression of GsCBRLK in transgenic Arabidopsis resulted in enhanced plant tolerance to high salinity and ABA and increased the expression pattern of a number of stress gene markers in response to ABA and high salt. These results identify GsCBRLK as a molecular link between the stress- and ABA-induced calcium/calmodulin signal and gene expression in plant cells.

Developmental Iodine Deficiency and Hypothyroidism Reduce Phosphorylation of Calcium/Calmodulin-Dependent Kinase II in the Rat Entorhinal Cortex

Iodine is essential for the synthesis of triiodothyronine (T₃) and thyroxine (T₄). Iodine deficiency leads to inadequate thyroid hormone. Hypothyroidism induced by iodine deficiency during gestation and postnatal period leads to cognitive deficits in learning and memory. However, the mechanism underlying these deficits is unclear. Calcium-dependent calmodulin kinase II (CaMKII) known as a potential memory molecule regulates important neuronal functions including learning and memory. Recent studies have shown that hypothyroidism alters phosphorylation of CaMKII in hippocampus or even in sympathetic ganglia of rats. Though the entorhinal cortex (EC) is an important functional structure within the neuronal network responsible for learning and memory, little is known about the effect of hypothyroidism on phosphorylation of CaMKII in the EC. Here, we report that iodine deficiency and propylthiouracil treatment through gestation and lactation reduce phosphorylation of CaMKII in the EC of pups. The increase of calcineurin, as well as reduction of neurogranin and calmodulin, may account for the reduced phosphorylation of CaMKII induced by developmental iodine deficiency and hypothyroidism. These findings in the EC may contribute to understanding the mechanisms that underlie impairment of learning and memory induced by developmental iodine deficiency and hypothyroidism.

HBx targeting to mitochondria and ROS generation are necessary but insufficient for HBV-induced cyclooxygenase-2 expression

Chronic inflammation can be a major risk factor for cancer development and may contribute to the high worldwide incidence of hepatocellular carcinoma (HCC). Cyclooxygenase-2 (COX-2) is known to be an important mediator of inflammatory responses; however, its link to hepatitis B virus (HBV)-mediated inflammatory responses has not been established. Here, we demonstrate that the expression of COX-2 mRNA and protein was significantly elevated in cells transfected by HBV replicon but not in cells transfected by HBV genome lacking the HBx gene. Notably, COX-2 induction was correlated with HBx's ability to increase reactive oxygen species (ROS) levels. Consistently with this, antioxidant treatment and ectopic expression of manganese superoxide dismutase or catalase completely abolished COX-2 induction. Interestingly, a mitochondria localization-defective mutant of HBx (HBx(Delta 68-117)) neither increased intracellular ROS levels nor induced COX-2 expression. HBx(68-117), which encodes only amino acids 68-117 and is sufficient for mitochondria localization, increased ROS levels but did not induce COX-2 expression. Similarly, HBx targeting to the outer membrane of mitochondria (Mito-HBx) increased ROS but also failed to increase COX-2 expression, suggesting that other cytoplasmic signaling pathways are involved in HBx-mediated COX-2 induction. Indeed, inhibition of cytoplasmic calcium signaling by cyclosporine A, blocking mitochondrial permeability transition pore, and herbimycin, and inhibition of calcium-dependent tyrosine kinase suppressed HBV-mediated COX-2 induction. Thus, the data indicate that both mitochondrial ROS and cytoplasmic calcium signaling are necessary for the COX-2 induction. Our studies revealed a pathophysiological link between HBV infection and hepatic inflammation, and this chain of events might contribute to early steps in HBV-associated liver carcinogenesis.

Dopamine Agonist Signalling in the Hypothalamus of Female Rats is Independent of Calcium‐Dependent Kinases

We have previously demonstrated that dopamine agonist, SKF38396 (SKF), can substitute for progesterone in the facilitation of female reproductive behaviour in oestradiol benzoate-primed female rats and mice. We also reported that both progesterone- and SKF-initiated signalling were mediated by the cAMP-dependent protein kinase A signal transduction cascade. As the rapid effects of progesterone are also mediated by calcium-dependent kinases, calcium- and calmodulin-dependent kinase (CaMKII) and protein kinase (PKC), we sought to determine whether SKF-initiated signalling also recruited calcium as a second messenger. We measured the changes in the activation of CaMKII and PKC in the ventromedial nucleus (VMN) of the hypothalamus and preoptic area (POA) of the rat brain, which are the two regions implicated in the regulation of female reproductive behaviour in rodents. We measured the basal activities representing the activation of the kinases by in vivo treatments, as well as the total kinase activities assayed in the presence of exogenous cofactors in vitro. We report that, in contrast to progesterone-initiated signalling, there was no recruitment of calcium by SKF in the hypothalamus, as shown by the absence of changes in CaMKII activities in the VMN and POA. Furthermore, SKF-treatment resulted in a rapid increase in calcium-independent basal PKC activity in the VMN but not the POA. These rapid changes were not the result of changes in PKC protein levels or phosphorylation status. These data indicate that progesterone- and SKF-recruit distinct signalling molecules within the same regions of the brain to activate region-specific signal transduction pathways.

Involvement of mitochondrial permeability transition in hepatitis B virus replication

The HBx protein of human hepatitis B virus (HBV) activates a calcium-dependent kinase pathway which is essential for the viral replication. In this study, we found that HBx expression in the absence of other HBV proteins and in the context of HBV replication decreased the mitochondrial calcein-AM/CoCl 2 signals by 10% and 14% in HepG2 cells and by 15% and 10% in Huh7 cells, respectively. This indicates that HBx can induce mitochondrial permeability transition (MPT) and cause calcium effusion into the plasma. In addition, RNA interference of Cylophilin D decreased HBx-induced MPT and suppressed HBV DNA replication by 41% in HepG2 cells. Our results suggest that HBx expression can induce MPT and facilitate HBV DNA replication.

Specific Detection of CD56 (NCAM) Isoforms for the Identification of Aggressive Malignant Neoplasms with Progressive Development

Alternative splicing of transcripts from many cancer-associated genes is believed to play a major role in carcinogenesis as well as in tumor progression. Alternative splicing of one such gene, the neural cell adhesion molecule CD56 (NCAM), impacts the progression, inadequate therapeutic response, and reduced total survival of patients who suffer from numerous malignant neoplasms. Although previous investigations have determined that CD56 exists in three major isoforms (CD56(120kD), CD56(140kD), and CD56(180kD)) with individual structural and functional properties, neither the expression profiles nor the functional relevance of these isoforms in malignant tumors have been consistently investigated. Using new quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) strategies and novel CD56 isoform-specific antibodies, CD56(140kD) was shown to be exclusively expressed in a number of highly malignant CD56(+) neoplasms and was associated with the progression of CD56(+) precursor lesions of unclear malignant potential. Moreover, only CD56(140kD) induced antiapoptotic/proliferative pathways and specifically phosphorylated calcium-dependent kinases that are relevant for tumorigenesis. We conclude, therefore, that the specific detection of CD56 isoforms will help to elucidate their individual functions in the pathogenesis and progression of malignant neoplasms and may have a positive impact on the development of CD56-based immunotherapeutic strategies.

Toxoplasma PRP1 is an ortholog of parafusin (PFUS) in vesicle scaffold assembly in Ca 2+-regulated exocytosis

The Paramecium tetraurelia protein parafusin (PFUS) and the Toxoplasma gondii protein parafusin-related protein 1 (PRP1) both have two covalent modifications (phosphorylation and phosphoglucosylation) and both are members of the phosphoglucomutase superfamily, associating with secretory vesicle scaffolds in their respective cells. This study tests the hypothesis that PRP1 is a functional ortholog of PFUS, functioning identically in Ca 2+-regulated exocytosis. Electroporation of fluorescently labeled recombinant His-PRP1 into live Paramecium cells resulted in its localization to docked, dense-core secretory vesicles (DCSVs) in a pattern identical to endogenous PFUS. In tam8 mutants, defective in transport of DCSVs, the fluorescently labeled protein was restricted to the un-transported DCSVs. Specificity of PRP1 localization was demonstrated by electroporating labeled actin or pyruvate kinase, which both failed to localize to either docked or undocked vesicles. In wild-type Paramecium, electroporated His-PRP1 dissociated from DCSVs upon exocytosis, and re-associated as new organelles formed. Mutagenized His-PRP1 species (S146A or S146E) cannot be phosphorylated by P2 calcium-dependent kinase in vitro. Upon electroporation, these molecules remained cytoplasmic and un-associated with DCSVs, while mutation of another PRP1 serine residue (S560A) did neither affect the localization to the DCSVs nor the phosphorylation pattern. Therefore, in this heterologous system, localization, transport and dissociation/re-association of PRP1 substituted for PFUS, supporting the conclusion that the proteins are functional orthologs. The assay also identified a strategic residue S146 within the PFUS ortholog (S138 in PFUS by extrapolation) required for post-translational modification, DCSV scaffold association and for exocytosis.

Cloning and characterization of a calcium dependent protein kinase gene associated with cotton fiber development

The gene GhCPK1 encoding a calcium dependent protein kinase was identified from cotton. Transcripts of GhCPK1 accumulated primarily in the elongating fiber, and Arabidopsis plants transformed with GhCPK1 promoter-GUS construct exhibited GUS activity mainly in the developing trichomes, roots, young leaves and sepals. In the bombarded onion epidermal cells, GhCPK1-GFP fusion proteins showed a subcellular distribution in the plasma membrane. In vitro assays indicated that GhCPK1 was a functional calcium-dependent kinase able to undergo autophosphorylation and phosphorylation of the known substrate histone III-S. Together, these results suggest that GhCPK1 may play a role in the calcium signaling events associated with fiber elongation.

Maize Plasma Membrane Aquaporins Belonging to the PIP1 and PIP2 Subgroups are in vivo Phosphorylated

Aquaporins are channel proteins that facilitate transmembrane water movement. In this study, we showed that plasma membrane intrinsic proteins (PIPs) from maize shoots are in vitro and in vivo phosphorylated on serine residues by a calcium-dependent kinase associated with the membrane fraction. Mass spectrometry identified phosphorylated peptides corresponding to the C-terminal region of (i) ZmPIP2;1, ZmPIP2;2 and/or ZmPIP2;7; (ii) ZmPIP2;3 and/or ZmPIP2;4; (iii) ZmPIP2;6; together with (iv) a phosphorylated peptide located in the N-terminal region of ZmPIP1;1, ZmPIP1;2, ZmPIP1;3 and/or ZmPIP1;4. The role of phosphorylation in the water channel activity of wild-type and mutant ZmPIP2;1 was studied in Xenopus laevis oocytes. Activation of endogenous protein kinase A increased the osmotic water permeability coefficient of ZmPIP2;1-expressing oocytes, suggesting that phosphorylation activates its channel activity. Mutation of S126 or S203, putative phosphorylated serine residues conserved in all plant PIPs, to alanine decreased ZmPIP2;1 activity by 30-50%, without affecting its targeting to the plasma membrane. Mutation of S285, which is phosphorylated in planta, to alanine or glutamate did not affect the water channel activity. These results indicate that, in oocytes, S126 and S203 play an important role in ZmPIP2;1 activity and that phosphorylation of S285 is not required for its activity.

Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB

Glutamate is a critical neurotransmitter of the central nervous system (CNS) and also an important regulator of cell survival and proliferation. The binding of glutamate to metabotropic glutamate receptors induces signal transduction cascades that lead to gene-specific transcription. The transcription factor NF-kappaB, which regulates cell proliferation and survival, is activated by glutamate; however, the glutamate receptor-induced signaling pathways that lead to this activation are not clearly defined. Here we investigate the glutamate-induced activation of NF-kappaB in glial cells of the CNS, including primary astrocytes. We show that glutamate induces phosphorylation, nuclear accumulation, DNA binding, and transcriptional activation function of glial p65. The glutamate-induced activation of NF-kappaB requires calcium-dependent IkappaB kinase alpha (IKKalpha) and IKKbeta activation and induces p65-IkappaBalpha dissociation in the absence of IkappaBalpha phosphorylation or degradation. Moreover, glutamate-induced IKK preferentially targets the phosphorylation of p65 but not IkappaBalpha. Finally, we show that the ability of glutamate to activate NF-kappaB requires cross-coupled signaling with the epidermal growth factor receptor. Our results provide insight into a glutamate-induced regulatory pathway distinct from that described for cytokine-induced NF-kappaB activation and have important implications with regard to both normal glial cell physiology and pathogenesis.

Pyk2 mediates increased adrenergic contractile responses in arteries from DOCA-salt mice — Vasoactive Peptide Symposium

The calcium-dependent proline-rich tyrosine kinase (Pyk2), a nonreceptor protein activated by tyrosine phosphorylation, links G protein-coupled receptors to vascular responses. We tested the hypothesis that enhanced vascular reactivity in deoxycorticosterone acetate (DOCA)-salt hypertensive mice is due to increased activation of Pyk2. Aorta and small mesenteric arteries from DOCA-salt and uninephrectomized (UNI) male C57Bl/6 mice were used. Systolic blood pressure (mm Hg) was higher in DOCA (126 ± 3) vs. UNI (100 ± 4) mice. Vascular responses to phenylephrine (1 nM to 100 μM) were greater both in aorta and small mesenteric arteries from DOCA-salt than UNI, but treatment with Tyrphostin A-9 (0.1 μM, Pyk2 inhibitor) abolished the difference among the groups. Pyk2 levels, as well as phospho-Pyk2 Tyr402, paxillin, and phospho-paxillin Tyr118 were increased in DOCA-salt aorta. Incubation of vessels with Tyrphostin A-9 restored phosphorylation of Pyk2 and paxillin. Increased activation of Pyk2 contributes to increased vascular contractile responses in DOCA-salt mice.

Calcium Signals Prime Macrophages

Interferon-α (IFN-α) stimulates macrophages to produce an inflammatory response. Wang et al . show that the activation of signal transducer and activator of transcription 1 (STAT1), but not STAT2, downstream of the IFN-α receptor (IFNAR) is regulated by crosstalk from signals generated by immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as integrins (mediating cell adhesion) and CCR5 (a G protein-coupled receptor for the chemokines CCL3, CCL4, and CCL5). STAT activation requires tyrosine phosphorylation, which then allows STAT proteins to dimerize and translocate to the nucleus to regulate gene expression. Wang et al . found basal phosphorylation of STAT1 on Ser 727 in unstimulated primary human macrophages. Ser 727 phosphorylation, as well as tyrosine phosphorylation (Tyr 701 ) stimulated by IFN-α, was inhibited by the calcium/calmodulin kinase (CaMK) inhibitor KN-93, the calmodulin inhibitor W-7, or the calcium chelator BAPTA. Thus, a basal calcium signal appears to be involved in this process of sensitizing cells to respond to IFN-α stimulation with STAT1 tyrosine phosphorylation. Although CaMK activity was likely to regulate STAT1 Ser 727 phosphorylation, it was not directly responsible for the tyrosine phosphorylation. Instead the calcium-dependent tyrosine kinase Pyk2 was found to interact with the tyrosine kinases associated with the IFNAR, Jak1, and Tyk2, and a role for Pyk2 in enhancing IFN-α-stimulated STAT1 tyrosine phosphorylation was confirmed by experiments with pharmacological inhibitors, cells expressing dominant-negative mutants, cells in which Pyk2 was depleted, and Pyk2-knockout cells. STAT2 activation was not affected by the treatments that altered STAT1 activation, suggesting that these two STATs may have different sensitivities to IFN-α. Dose-response experiments with a Jak1 inhibitor indicated that phosphorylation of STAT2 required low Jak1 activity, whereas STAT1 tyrosine phosphorylation required higher Jak1 activity. Thus, the basal calcium signal may help boost the STAT1 activation in response to low IFN-α signaling. The IFNAR does not produce a calcium signal; however, receptors that couple to the ITAM adaptor DAP12, which is basally active in macrophages, do stimulate calcium signals. At least two receptors that couple to DAP12 appear to contribute to the basal calcium signal and priming of the STAT1 response: integrins and CCR5. In experiments with mouse bone marrow macrophages, which are more adherent than the primary human macrophages from blood, detachment of the cells from the substrate decreased Pyk2 phosphorylation and decreased STAT1 activation in response to IFN-α. Primary human macrophages were found to produce ligand for the CCR5 receptor, and blocking CCR5 activity with a function-blocking antibody decreased CaMK activation and decreased STAT1 activation in response to IFN-α. Finally, in a mouse model of systemic lupus erythematosus, STAT1 was basally phosphorylated at Ser 727 in cells from the kidney (an affected organ in the mouse model). When the mice were treated with IFN-α, the kidney cells exhibited increased STAT1 activation compared with that in cells from control mice. The enhanced STAT1 activation and the basal Ser 727 phosphorylation were reduced in mice treated with the CaMK inhibitor KN-93. Thus, crosstalk through receptors coupled to DAP12 contributes a signal that tunes the IFN-α response and may be amenable to therapeutic intervention for treatment of autoimmune disease. L. Wang, I. Tassiulas, K.-H. Park-Min, A. C. Reid, H. Gil-Henn, J. Schlessinger, R. Baron, J. J. Zhang, L. B. Ivashkiv, 'Tuning' of type I interferon-induced Jak-STAT1 signaling by calcium-dependent kinases in macrophages. Nat. Immunol. 9 , 186-193 (2008). [PubMed]

Folate deprivation increases tau phosphorylation by homocysteine-induced calcium influx and by inhibition of phosphatase activity: Alleviation by S-adenosyl methionine

Several recent studies have indicated that increased levels of homocysteine (HC), including that resulting from deficiency in folate, increases tau phosphorylation. Some studies indicate that this is accomplished via HC-dependent activation of NMDA channels and resultant activation of calcium-dependent kinase pathways, while others suggest that the increase in tau phosphorylation is derived via HC-dependent inhibition of methylation of phosphatases and resultant inhibition of phosphatase activity. We demonstrate herein in SH-SY-5Y human neuroblastoma that both of these phenomena contribute to the increase in phospho-tau immunoreactivity following folate deprivation, and that supplementation with S-adenosyl methionine (SAM) prevents both the increase in kinase activity and the decrease in phosphatase activity. These findings demonstrate that the divergent neuropathological consequences of folate deprivation includes multiple pathways that converge upon tau phosphorylation, and further support the notion that dietary supplementation with SAM may reduce or delay neurodegeneration.

Reactive oxygen species mediate ERK activation through different Raf-1-dependent signaling pathways following cerebral ischemia

Production of reactive oxygen species (ROS) results in up-regulation of the extracellular signal-regulated kinase (ERK) cascade in response to numerous stimuli. Cerebral ischemia induces calcium-dependent kinase activation followed by ROS production. Here, we examined how ROS mediates the activation of ERK following cerebral ischemia in the rat hippocampus. We found that alpha-tocopherol, a free radical scavenger, attenuated the initial, robust activation of ERK by inhibiting Raf-1 dephosphorylation at Ser259. Alpha-tocopherol also down-regulated the second and mild activation of ERK through inhibition of Src-dependent phosphorylation of Raf-1 at Tyr340/341. Our results suggest that ROS production mediates the biphasic activation of ERK through different signaling cascades following post-ischemic reperfusion. Mediation of these signaling pathways involves changes in Raf-1 phosphorylation at different sites.

'Tuning' of type I interferon–induced Jak-STAT1 signaling by calcium-dependent kinases in macrophages

Immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors modulate the amplitude and nature of macrophage responses to Toll-like receptor and cytokine receptor stimulation. However, the molecular mechanisms enabling this receptor crosstalk are not known. Here we investigated the function of the calcium-dependent kinases CaMK and Pyk2 'downstream' of ITAM-associated receptors in the regulation of cytokine-induced activation of Jak kinases and STAT transcription factors. CaMK and Pyk2 relayed signals from integrins and the ITAM-containing adaptor DAP12 to augment interleukin 10- and interferon-alpha-induced Jak activation and STAT1-dependent gene expression. CaMK inhibition suppressed STAT1-mediated interferon-alpha signaling in a mouse model of systemic lupus erythematosus. Our results associate Pyk2 and Jak kinases with the linkage of signals emanating from cytokine and heterologous ITAM-dependent receptors.

CPI-17 Contributes to Calcium-Independent ADP-Induced Platelet Shape Change through P2Y1-Gq-PKC Pathways.

The initial event in platelet activation is the reorganization of the cytoskeleton causing the platelets to change from a discoid to a spiculated spheroid shape. Platelet shape change is primarily regulated by the phosphorylation of myosin light chain kinase. We have shown that this process is mediated through both calcium-dependent and calcium-independent Rho kinase pathways. CPI-17, a Protein kinase C (PKC) phosphorylated inhibitory protein of myosin light chain phosphatase, has been shown to have a role in platelet shape change downstream of thrombin-induced platelet activation. CPI-17 is a 17 kDa protein expressed in human platelets shown to inhibit myosin light chain phosphotase activity via PKCs. In this study we examined the role of CPI-17 in ADP-induced shape change and phosphorylation of CPI-17, downstream of the Gq coupled, P2Y1, and the Gi coupled, P2Y12 receptors. CPI-17 phosphorylation occurred upon activation of platelets with 2MeSADP. This phosphorylation was abolished in the presence of the P2Y1 receptor antagonist, MRS-2179. These results indicated that Gq signaling is important for platelet shape change and phosphorylation of CPI-17. In the presence of the calcium chelator, BAPTA, platelets changed shape in response to 2MeSADP; CPI-17 phosphorylation, however, was unaffected by BAPTA treatment under these conditions. However, CPI-17 phosphorylation was inhibited in the presence of the pan PKC inhibitors. These results indicate that CPI-17 phosphorylation occurs downstream of PKC activation. In the presence of BAPTA, treatment with PKC inhibitors decreased platelet shape change possibly due to reduced CPI-17 phosphorylation. The shape change caused by p160ROCK downstream of G12/13 pathways was unaffected by pan PKC inhibitors, but abolished by p160ROCK inhibitors H1152 or Y27632. Platelets incubated with BAPTA, pan PKC inhibitors, and p160ROCK inhibitor H1152, abolished ADP-induced platelet shape change and CPI-17 phosphorylation. In conclusion, ADP-induced platelet shape change occurs through a Gq-mediated, calcium-independent signaling pathway regulated by CPI-17 phosphorylation via PKC activation.

Abstract 1446: Central Role of Calcium-Dependent Tyrosine Kinase PYK2 in eNOS-Mediated Vascular Function and Angiogenic Response -Unmasked by PYK2 Knockout Mice-

Background: Endothelial nitric oxide synthase (eNOS) is activated by PI3-kinase/Akt or Ca 2+ -dependent CaM kinases, whereas the involvement of Ca 2+ -dependent tyrosine kinase PYK2 remains to be determined. Methods and Results: We newly generated the PYK2-deficient (−/−) mice and investigated the effects of PYK2 on NO-mediated vascular functions and angiogenic response. The PYK2−/− mice exhibited a significant reduction in the basal eNOS phosphorylation and VEGF- or Acetylcholin-mediated response in the aorta and aortic endothelial cells (ECs) compared with the wild-type mice, and blood flow recovery and neovessel formation after hindlimb ischemia were reduced with affecting mobilization of endothelial progenitors. Acetylcholin-mediated aortic vasorelaxation and cGMP production were decreased (54% and 44%, P<0.01, respectively) and blood pressure in the PYK2−/− mice is higher than the wild-type mice (mean 97 vs. 86 mmHg, P<0.05). In the PYK2−/− mice, VEGF-mediated Akt phosphorylation, NO production and cytoplasmic Ca 2+ mobilization were markedly decreased. And also the phosphorylation of c-Src and PLCγ1, interaction of c-Src/PLCγ1 were clearly reduced. VEGF-dependent migration, tube formation and actin cytoskeletal reorganization were markedly inhibited in PYK2−/− ECs, while addition of L-arginine or cGMP-dependent kinase activator reversed the attenuated response. Conclusion: These findings demonstrate that PYK2 is involved in the receptor-activated signaling events leading to the phosphorylation of eNOS and thus regulates eNOS-mediated vasoactive function and angiogenesis.