Activation of hematopoietic progenitor kinase 1 involves relocation, autophosphorylation, and transphosphorylation by protein kinase D1

Adaptive immune signaling can be coupled to stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and NF-kappaB activation by the hematopoietic progenitor kinase 1 (HPK1), a mammalian hematopoiesis-specific Ste20 kinase. To gain insight into the regulation of leukocyte signal transduction, we investigated the molecular details of HPK1 activation. Here we demonstrate the capacity of the Src family kinase Lck and the SLP-76 family adaptor protein Clnk (cytokine-dependent hematopoietic cell linker) to induce HPK1 tyrosine phosphorylation and relocation to the plasma membrane, which in lymphocytes results in recruitment of HPK1 to the contact site of antigen-presenting cell (APC)-T-cell conjugates. Relocation and clustering of HPK1 cause its enzymatic activation, which is accompanied by phosphorylation of regulatory sites in the HPK1 kinase activation loop. We show that full activation of HPK1 is dependent on autophosphorylation of threonine 165 and phosphorylation of serine 171, which is a target site for protein kinase D (PKD) in vitro. Upon T-cell receptor stimulation, PKD robustly augments HPK1 kinase activity in Jurkat T cells and enhances HPK1-driven SAPK/JNK and NF-kappaB activation; conversely, antisense down-regulation of PKD results in reduced HPK1 activity. Thus, activation of major lymphocyte signaling pathways via HPK1 involves (i) relocation, (ii) autophosphorylation, and (iii) transphosphorylation of HPK1 by PKD.     

A novel protein kinase C target site in protein kinase D is phosphorylated in response to signals for cardiac hypertrophy

Protein kinase D (PKD) regulates cardiac myocyte growth and contractility through phosphorylation of proteins such as class IIa histone deacetylases (HDACs) and troponin I (TnI). In response to agonists that activate G-protein-coupled receptors (GPCRs), PKD is phosphorylated by protein kinase C (PKC) on two serine residues (Ser-738 and Ser-742 in human PKD1) within an activation loop of the catalytic domain, resulting in stimulation of PKD activity. Here, we identify a novel PKC target site located adjacent to the auto-inhibitory pleckstrin homology (PH) domain in PKD. This site (Ser-412 in human PKD1) is conserved in each of the three PKD family members and is efficiently phosphorylated by multiple PKC isozymes in vitro. Employing a novel anti-phospho-Ser-412-specific antibody, we demonstrate that this site in PKD is rapidly phosphorylated in primary cardiac myocytes exposed to hypertrophic agonists, including norepinephrine (NE) and endothelin-1 (ET-1). Differential sensitivity of this event to pharmacological inhibitors of PKC, and data from in vitro enzymatic assays, suggest a predominant role for PKCδ in the control of PKD Ser-412 phosphorylation. Together, these data suggest a novel, signal-dependent mechanism for controlling PKD function in cardiac myocytes.     

Activation of a cytosolic serine protein kinase by epidermal growth factor

Exposure of A-431 cells to epidermal growth factor (EGF) results in a rapid enhancement (approximately 10-fold) of cytosolic serine protein kinase activity. The increase in serine kinase activity may be detected using a number of peptide and protein substrates. Enhancement of kinase activity occurs within 1 min of exposure of the cells to EGF and reaches a maximum in 5 min. Similar results were obtained with a variety of cell lines. We have partially purified the EGF-activated kinase from A-431 cells. It has an apparent molecular mass of approximately 100 kDa by gel filtration. One distinguishing property of the enzyme is its sensitivity to inhibition by micromolar quantities of polyarginine; polylysine has no effect. The EGF-activated kinase is unaffected by cyclic nucleotides, Ca2+/calmodulin, Ca2+/diolein/phosphatidylserine, or heparin. The enhancement of cytosolic serine kinase activity in A-431 cells appears to be an early event in cell "activation" by a number of biological response modifiers including EGF, bradykinin, 12-O-tetradecanoylphorbol-13-acetate, and histamine.     

Activation of MEK kinase 1 by the c-Abl protein tyrosine kinase in response to DNA damage

The c-Abl protein tyrosine kinase is activated by certain DNA-damaging agents and regulates induction of the stress-activated c-Jun N-terminal protein kinase (SAPK). Here we show that nuclear c-Abl associates with MEK kinase 1 (MEKK-1), an upstream effector of the SEK1-->SAPK pathway, in the response of cells to genotoxic stress. The results demonstrate that the nuclear c-Abl binds to MEKK-1 and that c-Abl phosphorylates MEKK-1 in vitro and in vivo. Transient-transfection studies with wild-type and kinase-inactive c-Abl demonstrate c-Abl kinase-dependent activation of MEKK-1. Moreover, c-Abl activates MEKK-1 in vitro and in response to DNA damage. The results also demonstrate that c-Abl induces MEKK-1-mediated phosphorylation and activation of SEK1-SAPK in coupled kinase assays. These findings indicate that c-Abl functions upstream of MEKK-1-dependent activation of SAPK in the response to genotoxic stress.     

IL-10-conditioned dendritic cells, decommissioned for recruitment of adaptive immunity, elicit innate inflammatory gene products in response to danger signals

Dendritic cells (DCs) are the professional APCs of the immune system, enabling T cells to perceive and respond appropriately to potentially dangerous microbes, while also being able to maintain T cell tolerance toward self. In part, such tolerance can be determined by IL-10 released from certain types of regulatory T cells. IL-10 has previously been shown to render DCs unable to activate T cells and it has been assumed that this process represents a general block in maturation. Using serial analysis of gene expression, we show that IL-10 pretreatment of murine bone marrow-derived DCs alone causes significant changes in gene expression. Furthermore, these cells retain the ability to respond to Toll-like receptor agonists, but in a manner skewed toward the selective induction of mediators known to enhance local inflammation and innate immunity, among which we highlight a novel CXCR2 ligand, DC inflammatory protein-1. These data suggest that, while the presence of a protolerogenic and purportedly anti-inflammatory agent such as IL-10 precludes DCs from acquiring their potential as initiators of adaptive immunity, their ability to act as initiators of innate immunity in response to Toll-like receptor signaling is enhanced.     

Activation of phosphoinositide hydrolysis by nerve growth factor and lysophosphatidylserine in rat peritoneal mast cells

Histamine secretion in rat peritoneal mast cells stimulated by nerve growth factor requires a synergistic signal delivered by lysophosphatidylserine. To study the signal-transducing system activated by these compounds, phospholipid metabolism has been investigated in these cells. Phospholipid labeling with 32PO4 reveals a 5-9-fold stimulation of phosphatidic acid, phosphatidylinositol and phosphatidylcholine synthesis. Increased synthesis of phosphatidylinositol is also monitored using [3H]inositol incorporation. When [3H]inositol-labeled mast cells are incubated in the presence of Li+, nerve growth factor and lysophosphatidylserine enhance the accumulation of inositol monophosphate, inositol bisphosphate and inositol trisphosphate. Similar to the induced histamine release, accumulation of inositol phosphates (a) does not occur when the two agonists are added separately; (b) is inhibited when lysophosphatidyl-L-serine is replaced by lysophosphatidyl-D-serine; and (c) is enhanced in the presence of extracellular Ca2+. The data suggest that the interactive stimulus of nerve growth factor and lysophosphatidylserine is transmitted through the polyphosphoinositide-phospholipase C system.     

Characterization of calcium-activated, phospholipid-dependent protein kinase from rat serosal mast cells and RBL-1 cells

Evidence is presented that rat serosal mast cells and cells of the rat basophilic leukemia line, RBL-1, each contain a calcium-activated, phospholipid-dependent protein kinase. The enzymes are very similar in their activation requirements to the calcium-dependent enzymes termed protein kinase Cs in brain. The enzyme is selectively stimulated by diolein and phosphatidylserine and is inhibited by several local anesthetics. The Ka for Ca2+ is 1.0 X 10(-3) M and 1.5 X 10(-4) M in mast cells and RBL-1 cells, respectively. The enzyme in mast cells is rapidly activated and apparently changed in its intracellular distribution when intact mast cells are stimulated with 48/80, A-23187, and anti-IgE and 12-O-tetradecanoyl-13-acetate in combination.     

Antigen-induced phospholipase D activation in rat mast cells is independent of protein kinase C

In the present study, we investigated the involvement of protein kinase C (PKC) in antigen (Ag, DNP-Ascaris suum)-induced phospholipase D (PLD) activation of rat peritoneal mast cells. Phorbor myristate acetate (PMA) as well as Ag activated PLD as inferred by phosphatidylethanol (PEt) production. PKC inhibitors, staurosporine and H-7, however, failed to suppress PMA-stimulated PLD activation, suggesting that PLD activation by PMA is independent of PKC. By contrast, Ag-stimulated PLD activity was significantly reduced by staurosporine and slightly by H-7. Surprisingly, the inhibitors inhibited Ag-stimulated phospholipase C (PLC), correlated to the inhibition of PLD. These observations lead us to conclude that in Ag-stimulated mast cells 1,2-diacylglycerol (DG) formed by PLC directly or indirectly stimulates PLD, independently of PKC.     

Activation of protein kinase C inhibits the expression of connective tissue growth factor

Connective tissue growth factor (CTGF) is a member of a protein family in which 38 cysteine residues are conserved. Although a wide variety of important biological functions have been ascribed to these proteins in recent years, the regulation of their gene expression for most members is virtually unknown. We studied the effects of protein kinase C (PKC) and tyrosine kinase on the expression of CTGF and observed that at the mRNA level CTGF expression is inhibited by the activation of PKC, but stimulated by the inhibition of PKC and tyrosine kinase. We further determined that the novel and the classical PKC isoforms are needed for the inhibition, but the atypical isoforms are not involved. Our data suggest that phosphorylation on serine/threonine and tyrosine by PKC and by tyrosine kinase are all inhibitory to the expression of CTGF.     

Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.     

NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase,regulates innate immunity and development in plants

Mitogen-activated protein kinase (MAPK) cascades are rapidly activated upon plant recognition of invading pathogens. Here, we describe the use of virus-induced gene silencing (VIGS) to study the role of candidate plant MAP kinase kinase kinase (MAPKKK) homologs of human MEKK1 in pathogen-resistance pathways. We demonstrate that silencing expression of a tobacco MAPKKK, Nicotiana Protein Kinase 1 (NPK1), interferes with the function of the disease-resistance genes N, Bs2, and Rx, but does not affect Pto- and Cf4-mediated resistance. Further, NPK1-silenced plants also exhibit reduced cell size, defective cytokinesis, and an overall dwarf phenotype. Our results provide evidence that NPK1 functions in the regulation of N-, Bs2-, and Rx-mediated resistance responses and may play a role in one or more MAPK cascades, regulating multiple cellular processes.     

Protein kinase C-alpha and protein kinase C-epsilon are required for Grb2-associated binder-1 tyrosine phosphorylation in response to platelet-derived growth factor

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.     

Rheb interacts with Raf-1 kinase and may function to integrate growth factor- and protein kinase A-dependent signals.

Rheb is a recently described member of the Ras family that was originally identified as an immediate-early gene in brain but is also widely expressed in other tissues. Here we demonstrate that Rheb interacts with and appears to regulate Raf-1 kinase, an essential component of the H-Ras signaling pathway. In direct contrast to H-Ras, however, the interaction of Rheb with Raf-1 is potentiated by growth factors in combination with agents that increase cyclic AMP (cAMP) levels. Protein kinase A-dependent phosphorylation of serine 43 within the regulatory domain of Raf-1 reciprocally potentiates its interaction with Rheb and decreases its interaction with H-Ras. A single amino acid in the G2 effector domain is critical for the differential properties of Rheb. Since Rheb is an immediate-early gene, our studies suggest that Rheb functions in concert with H-Ras to dynamically integrate cAMP and growth factor signaling.     

Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochromocytoma cells

We have used a permeabilized cell assay and a synthetic peptide substrate (KRTLRR) to specifically monitor the activity of protein kinase C in PC12 cells preincubated with nerve growth factor (NGF), epidermal growth factor (EGF), or phorbol esters. Pretreatment of PC12 cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate or 1 microM phorbol dibutyrate stimulated the rate of KRTLRR peptide phosphorylation 4.8- and 2.6-fold, respectively. Furthermore, pretreatment of cells with NGF or EGF transiently increased the KRTLRR peptide kinase activity. Peak stimulations of KRTLRR peptide kinase (1.3-2-fold) were observed after 1-5 min of growth factor treatment and returned to control levels within 15-20 min. The KRTLRR peptide kinase activity fulfilled two criteria of protein kinase C. A synthetic peptide inhibitor of protein kinase C inhibited both growth factor- and phorbol ester-stimulated KRTLRR peptide kinase activity. In addition, growth factors and phorbol esters failed to stimulate KRTLRR peptide kinase activity in cells rendered protein kinase C-deficient by long-term treatment with 1 microM 12-O-tetradecanoylphorbol 13-acetate. In contrast to the transient activation of protein kinase C, ribosomal S6 kinase, assayed with the synthetic peptide RRLSSLRA, was persistently activated by NGF and EGF. The findings indicate that protein kinase C serves an early and transient role in the molecular actions of NGF and EGF in PC12 cells.     

Activation of Rac1 and the p38 mitogen-activated protein kinase pathway in response to arsenic trioxide

Arsenic trioxide induces differentiation and apoptosis of malignant cells in vitro and in vivo, but the mechanisms by which such effects occur have not been elucidated. In the present study we provide evidence that arsenic trioxide induces activation of the small G-protein Rac1 and the alpha and beta isoforms of the p38 mitogen-activated protein (MAP) kinase in several leukemia cell lines. Such activation of Rac1 and p38-isoforms results in downstream engagement of the MAP kinase-activated protein kinase-2 and is enhanced by pre-treatment of cells with ascorbic acid. Interestingly, pharmacological inhibition of p38 potentiates arsenic-dependent apoptosis and suppression of growth of leukemia cell lines, suggesting that this signaling cascade negatively regulates induction of antileukemic responses by arsenic trioxide. Consistent with this, overexpression of a dominant-negative p38 mutant (p38betaAGF) enhances the antiproliferative effects of arsenic trioxide on target cells. To further define the relevance of activation of the Rac1/p38 MAP kinase pathway in the induction of arsenic-dependent antileukemic effects, studies were performed using bone marrows from patients with chronic myelogenous leukemia. Arsenic trioxide suppressed the growth of leukemic myeloid (CFU-GM) progenitors from such patients, whereas concomitant pharmacological inhibition of the p38 pathway enhanced its growth-suppressive effects. Altogether, these data provide evidence for a novel function of the p38 MAP kinase pathway, acting as a negative regulator of arsenic trioxide-induced apoptosis and inhibition of malignant cell growth.     

Phosphatidylinositol 3-kinase and protein kinase D1 specifically cooperate to negatively regulate the insulin-like growth factor signaling pathway

Insulin receptor substrate-1 (IRS-1) is a key protein in the insulin-like growth factor (IGF) signaling whose tyrosine phosphorylation by the type 1 IGF receptor is necessary for the recruitment and activation of the downstream effectors. Through the analysis of cross-talks occurring between different tyrosine kinase receptor-dependent signaling pathways, we investigated how two growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] could modulate the IGF-I-induced IRS-1 tyrosine phosphorylation and its downstream signaling. EGF and FGF inhibited IGF-I-stimulated tyrosine phosphorylation of IRS-1 and the subsequent IGF-I-induced phosphatidylinositol 3-kinase (PI 3-kinase) activity. These EGF- and FGF-inhibitory effects were dependent on both PI 3-kinase and protein kinase D1 (PKD1) signaling pathways but independent on the extracellular signal-regulated kinase (ERK) pathway. PKD1, which was activated independently of the PI 3-kinase pathway, associated with IRS-1 in response to EGF or FGF. Unlike PI 3-kinase, PKD1 did not mediate the EGF- or FGF-induced-IRS-1 serine 307 phosphorylation which was described to inhibit IRS-1. Interestingly, specific inhibition of either PI 3-kinase or PKD1 totally impaired EGF- or FGF-induced inhibition of IGF-I-stimulated IRS-1 tyrosine phosphorylation. This indicated that serine 307 phosphorylation of IRS-1 is not sufficient per se to inhibit the IGF signaling pathway and demonstrated for the first time that the negative regulation of IRS-1 requires the coordinated action of PI 3-kinase and PKD1. This further suggests that PKD1 may be an attractive target for innovative strategies that target the IGF signaling pathway.     

Activation of the purified protein tyrosine kinase domain of the epidermal growth factor receptor

Biological responses to epidermal growth factor (EGF) depend on the ligand-stimulated protein tyrosine kinase activity of its receptor. To further characterize the enzymatic activity of the EGF receptor, the baculovirus expression system was used to express the cytoplasmic protein tyrosine kinase domain of the EGF receptor. Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus correctly expressed an active tyrosine kinase domain of the EGF receptor as demonstrated by 35S metabolic labeling, immunoblotting with anti-EGF receptor and anti-phosphotyrosine antibodies, and autophosphorylation analysis. The kinase domain (Mr 66,000) was purified to near homogeneity using a monoclonal anti-phosphotyrosine antibody column, providing 0.5 mg of kinase domain/liter of Sf9 cells (23% yield). The purified kinase domain exhibited a strong preference for Mn2+ compared to Mg2+. The specific activity of the kinase domain was low compared to purified, EGF-activated EGF receptor. However, the addition of sphingosine or ammonium sulfate greatly increased the activity of the kinase domain to equal or exceed the activity of ligand-activated holo EGF receptor. These results indicate that the addition of sphingosine or ammonium sulfate to the purified kinase domain can mimic the effect of EGF to induce a conformation of the holo EGF receptor which is optimal for tyrosine kinase activity. Deletion of the ligand binding domain, analogous to that which occurs in erb B, is not sufficient to fully activate the kinase, implying that EGF causes conformational changes additional to removal of an inhibitory constraint.