Assaying Bcr-Abl kinase activity and inhibition in whole cell extracts by phosphorylation of substrates immobilized on agarose beads

There is a current and increasing demand for simple, robust, nonradioactive assays of protein tyrosine kinase activity with applications for clinical diagnosis and high-throughput screening of potential molecularly targeted therapeutic agents. One significant challenge is to detect and measure the activity of specific kinases with key roles in cell signaling as an approach to distinguish normal cells from cancer cells and as a means of evaluating targeted drug efficacy and resistance in cancer cells. Here, we describe a method in which kinase substrates fused to glutathione-S-transferase and immobilized on glutathione agarose beads are phosphorylated, eluted, and then assayed to detect kinase activity. The activity of recombinant, purified c-Abl kinase or Bcr-Abl kinase in whole cell extracts can be detected with equivalent specificity, sensitivity, and reproducibility. Similarly, inhibition of recombinant c-Abl or Bcr-Abl in cells or cell extracts by imatinib mesylate and other Bcr-Abl targeted kinase inhibitors is readily assayed. This simple kinase assay is sufficiently straightforward and robust for use in clinical laboratories and is potentially adaptable to high-throughput assay formats.     

Protein-acrylamide copolymer hydrogels for array-based detection of tyrosine kinase activity from cell lysates

We describe the development of an array-based assay for the molecular level detection of tyrosine kinase activity directly from cellular extracts. Glutathione S-transferase-Crkl (GST-Crkl) fusion proteins are covalently immobilized into polyacrylamide gel pads via copolymerization of acrylic monomer and acrylic-functionalized GST-Crkl protein constructs on a polyacrylamide surface. The resulting hydrogels resist nonspecific protein adsorption, permitting quantitative and reproducible determination of Abl tyrosine kinase activity and inhibition, even in the presence of a complex cell lysate mixture. Half-maximal inhibition (IC50) values for imatinib mesylate inhibition of GST-Crkl (SH3) phosphorylation by v-Abl in a purified system and Bcr-Abl within a K562 cell lysate were determined to be 1.5 and 20 microM, respectively. Additionally, the protein-acrylamide copolymer arrays detected CML cell levels as low as 15% in a background of Bcr-Abl- leukemic cells and provided the framework for the parallel evaluation of six tyrosine kinase inhibitors. Such a system may have direct application to the detection and treatment of cancers resulting from upregulated tyrosine kinase activity, such as chronic myeloid leukemia (CML). These findings also establish a basis for screening tyrosine kinase inhibitors and provide a framework on which protein-protein interactions in other complex systems can be studied.     

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.     

Changes in the proteome associated with the action of Bcr-Abl tyrosine kinase are not related to transcriptional regulation

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease, the hallmark of which is the Bcr-Abl protein tyrosine kinase (PTK). Without intervention the disease progresses from a benign chronic phase to a rapidly fatal blast crisis. To identify the molecular mechanisms underlying disease progression we used two-dimensional gel electrophoresis on a model we have previously described using the expression of a conditional mutant of Bcr-Abl PTK in a multipotent stem cell line, FDCP-Mix. Long term exposure of FDCP-Mix cells to Bcr-Abl mimics disease progression in CML. Four major differences were observed as a consequence of long term exposure to the Bcr-Abl PTK compared with cells exposed short term. The proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry-generated peptide mass fingerprint data and liquid chromatography-tandem mass spectrometry-generated sequence information. Leukotriene A4 hydrolase, an enzyme known to be deregulated in CML, was found to be up-regulated. Annexin VI, vacuolar ATP synthase catalytic subunit A, and mortalin were found to be down-regulated. Poly(A) PCR cDNA analysis showed there was no correlation between the protein expression changes and mRNA levels. Western blot analysis also indicated no change in the levels of mortalin or leukotriene A4 hydrolase, indicating that post-translational events may modify protein content of the specific spots. Leukotriene B4 levels (product of leukotriene A4 hydrolase) were, however, reduced in cells exposed long term to Bcr-Abl activity. This study demonstrates the potential of proteomic analysis to define novel effects of oncogenes.     

The Marine-Derived Oligosaccharide Sulfate (MdOS), a Novel Multiple Tyrosine Kinase Inhibitor,Combats Tumor Angiogenesis both In Vitro and In Vivo

Despite the emerging success of multi-targeted protein tyrosine kinase (PTK) inhibitors in cancer therapy, significant side effects and resistance concerns seems to be avoided unlikely. The aim of the present study was to identify novel multi-targeting PTK inhibitors. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay (ELISA). The antiproliferative activities in human microvascular endothelial cells (HMECs) were evaluated by sulforhodamine (SRB) assay. The phosphorylation of kinases and their downstream molecules was probed by western blot analysis. The binding mode between MdOS and PTKs was profiled by surface plasmon resonance (SPR) approach and molecular simulation. Tube formation assay, rat aortic ring method and chicken chorioallantoic membrane assay were combined to illustrate the in vitro and in vivo anti-angiogenic effects. Results indicated that MdOS, a novel marine-derived oligosaccharide sulfate, exhibited a broad-spectrum PTK inhibitory action. At an enzymatic level, MdOS inhibited HER2, EGFR, VEGFR, PDGFR, c-Kit, FGFR1 and c-Src, with little impact on FGFR2. In cellular settings, MdOS inhibited phosphorylation of PTKs, exemplified by HER2, EGFR and VEGFR2, and downstream molecules of Erk1/2 and AKT. Further studies demonstrated that MdOS acted as an ATP-competitive inhibitor via directly binding to the residues of entrance rather than those of the ATP-binding pocket. Furthermore, MdOS inhibited proliferation and tube formation of HMECs, arrested microvessel outgrowth of rat aortic rings and hindered the neovascularization of chick allantoic membrane. Taken together, results presented here indicated that MdOS exhibited anti-angiogenic activity in a PTK-dependent manner and make it a promising agent for further evaluation in PTK-associated cancer therapy.     

A Multiple Reaction Monitoring (MRM) Method to Detect Bcr-Abl Kinase Activity in CML Using a Peptide Biosensor

The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1–5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient’s Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ∼15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.     

Modulation of monocytic differentiation of HL-60 cells by inhibitors of protein tyrosine kinases.

We showed previously that the expressions of various src family protein tyrosine kinases (PTKs) were induced independently during the monocytic differentiation of HL-60 cells. The role of PTKs was further assessed in the present study by investigating the effects of PTK inhibitors on the differentiation. It was demonstrated that PTK inhibitors such as genistein and herbimycin A modulated monocytic differentiation of HL-60 cells; they inhibited the differentiation induced by TPA, while promoting that induced by vitamin D3 (D3). Immunoblotting analysis of protein molecules which had been phosphorylated on their tyrosine residues demonstrated that TPA induced phosphorylation of certain molecules different from those induced by D3 in HL-60 cells. PTK inhibitors blocked the phosphorylation and modulated differentiation driven by the inducers. These data suggest that PTKs are involved both promotively and suppressively in signaling events that induce monocytic differentiation of HL-60 cells.     

Assessment of downstream effectors of BCR/ABL protein tyrosine kinase using combined proteomic approaches

Leukaemic transformation is frequently associated with the aberrant activity of a protein tyrosine kinase (PTK). As such it is of clinical relevance to be able to map the effects of these leukaemogenic PTKs on haemopoietic cells at the level of phosphorylation modulation. In this paradigm study we have employed a range of proteomic approaches to analyse the effects of one such PTK, BCR/ABL. We have employed phosphoproteome enrichment techniques allied to peptide and protein quantification to identify proteins and pathways involved in cellular transformation. Amongst the proteins shown to be regulated at the post‐translational level were cofilin, an actin‐severing protein thus linked to altered motility and Cbl an E3 ubiquitin ligase integrally linked to the control of tyrosine kinase signalling (regulated by 5 and 6 PTKs respectively). The major class of proteins identified however were molecular chaperones. We also showed that HSP90 phosphorylation is altered by BCR/ABL action and that HSP90 plays a crucial role in oncogene stability. Further investigation with another six leukaemogenic PTKs demonstrates that this HSP90 role in oncogene stability appears to be a common phenomenon in a range of leukaemias. This opens up the potential opportunity to treat different leukaemias with HSP90 inhibitors.     

Oleylamine-carbonyl-valinol inhibits auto-phosphorylation activity of native and T315I mutated Bcr-Abl, and exhibits selectivity towards oncogenic Bcr-Abl in SupB15 ALL cell lines

Chronic myeloid leukemia (CML) is characterized by the presence of p210^Bcr-Abl which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of CML. Despite high rates of clinical response, CML patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein kinase domain. Previously, we have identified oleic acid as the active component in the mushroom Daedalea gibbosa that inhibited the kinase activity of Bcr-Abl. Here, we report that the oleyl amine derivatives, S-1-(1-Hydroxymethyl-2-methyl-propyl)-3-octadec-9-enyl-urea [oleylaminocarbonyl-L-N-valinol,oroleylaminocarbonyl-S-2-isopropyl-N-ethanolamine,oleylamine-carbonyl-L-valinol] (cpd 6) and R-1-(1-Hydroxymethyl-2-methyl-propyl)-3-octadec-9-enyl-urea [oleylamineocarbonyl-D-N-valinol, oleylaminocarbonyl-R-2-isopropyl-N-ethanolamine, or oleylamine-carbonyl-D-valinol] (cpd 7), inhibited the activity of the native and T315I mutated Bcr-Abl. Furthermore, cpd 6 and 7 exhibited higher activity towards the oncogenic Bcr-Abl in comparison to native c-Abl in SupB15 Ph-positive ALL cell line.     

Antisense inhibition of Bcr-Abl/c-Abl synthesis promotes telomerase activity and upregulates tankyrase in human leukemia cells

Clinical studies in chronic myelogenous leukemia demonstrate that the overexpression of Bcr-Abl tyrosine kinase is usually accompanied by relatively low telomerase activity in the chronic phase, which reverts to a high activity in blast crisis. The present study was designed to investigate the cross-talk between both enzymes, using Bcr-Abl-positive K-562 and Bcr-Abl-negative Jurkat cell lines, treated with antisense oligodeoxyribonucleotides (ODNs) against Bcr-Abl/c-Abl mRNA. The decreased amount and enzyme activity of Bcr-Abl/c-Abl provoked telomerase activation in both cell lines. After short-term treatment with anti-Bcr-Abl/c-Abl ODNs (6 days), no variations in hTERT and phospho-hTERT were detected. The decreased amount of Bcr-Abl/c-Abl was accompanied by: alterations in telomeric associated proteins-overexpression of tankyrase and decreased amount of TRF1/Tin2, cell growth arrest of K-562 cells, reaching a plateau after 6 days treatment, and increased proliferating activity of Jurkat cells. No changes in telomere length were detected after short-term treatment. In contrast, after long-term treatment with anti-Bcr-Abl/c-Abl ODNs (36 days), a significant elongation of telomeres and enhancement of hTERT were established, accompanied by an increased proliferating activity of both cell lines. These data provide evidence that the inhibition of Bcr-Abl or c-Abl synthesis keeps a potential to restore or induce cell proliferation through telomere lengthening control and telomerase activation.     

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60^c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60^c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60^c-src, with a K_i of 24 M. This peptide inhibitor exhibited selectivity for pp60^c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal l-1-naphthylalanine - 2-Nal l-2-naphthylalanine - BOP benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - cAPK cyclic AMP-dependent protein kinase - DIEA diisopropylethylamine - EGFR epidermal growth factor receptor - Fmoc fluorenylmethoxycarbonyl - HOBt 1-hydroxybenzotriazole - MES 2-[N-morpholino]ethanesulfonic acid - PBS phosphate-buffered salts - pCl l-p-chlorophenylalanine - pF l-p-fluorophenylalanine - PTK protein tyrosine kinase - TLC thin-layer chromatography     

Label-free electrochemical measurement of protein tyrosine kinase activity and inhibition based on electro-catalyzed tyrosine signaling

A novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1) peptide, as a substrate of EGFR, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy)(3)(2+) (bpy=2,2'-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 1 UmL(-1). Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC(50) of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants K(i) were estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery.     

IL-2 signaling in human monocytes involves the phosphorylation and activation of p59hck

The activating properties of IL-2 and the structure of the IL-2R on human monocytes are well characterized. However, relatively little is known about the biochemical mechanisms involved in IL-2 signal transduction in these cells. We investigated the role of protein tyrosine kinases (PTKs) in the activation of monocytes by IL-2. Incubation of monocytes with the PTK inhibitor herbimycin A (HA) resulted in the dose-dependent suppression of IL-2-induced monocyte tumoricidal activity. This inhibition was rather potent, as a concentration of HA as low as 0.5 microM caused a complete abrogation of cytolytic activity. Furthermore, HA markedly suppressed the ability of IL-2 to induce IL-1 beta, TNF-alpha, IL-6, and IL-8 mRNA expression and protein secretion by monocytes. Anti-phosphotyrosine immunoblotting demonstrated that IL-2 induced a rapid and time-dependent increase in tyrosine phosphorylation of several cellular proteins of molecular masses ranging from 35 to 180 kDa. Interestingly, IL-2 caused a significant up-regulation of the constitutive levels of hck PTK mRNA and protein relative to medium-treated cells as well as an increase in p59hck tyrosine phosphorylation. Finally, we demonstrated by in vitro kinase assay that the specific activity of p59hck PTK was also induced by IL-2 in monocytes. Thus, these data show that the activation of PTKs is required for the triggering of monocyte effector and secretory functions by IL-2 and strongly suggest that p59hck is a key participant in IL-2 signaling in human monocytes.     

Noncatalytic Inhibition of Cyclic Nucleotide–gated Channels by Tyrosine Kinase Induced by Genistein

Rod photoreceptor cyclic nucleotide–gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.     

Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia

One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis.     

Activation of macrophages with N-formyl-methionyl-leucyl-phenylalanine: involvement of protein kinase C and tyrosine kinase

N-formyl-methionyl-leucyl-phenylalanine (fMLP) a potent chemotactic peptide stimulates immune responses by activating macrophages and other cells of the immune system. The present study reports inhibition of fMLP-induced activation of murine peritoneal and P388D-1 macrophage cell line by protein kinase C (PKC) inhibitors, H-7 and chelerythrine chloride. Similarly, tumoricidal activity was also downregulated by protein tyrosine kinase (PTK) inhibitors genestein and lavendustin A. Further, fMLP increased tyrosine phosphorylation of several proteins in murine macrophages, which were inhibited in presence of genestein and lavendustin A. These findings suggest the involvement of PKC and PTK in the activation of murine macrophages with fMLP.     

Protein Tyrosine Kinase-Mediated Potentiation of Currents from Cloned NMDA Receptors

Abstract: Although serine/threonine phosphorylation has been more commonly recognized as a mechanism to modulate the function of ion channels and receptors, tyrosine phosphorylation is under increasing scrutiny. An important subtype of glutamate receptor, the NMDA receptor, is shown to be regulated by insulin via protein tyrosine kinase (PTK). NMDA currents through cloned receptors are potentiated by insulin in a subunit-specific manner. The insulin-mediated potentiation of NMDA current is diminished by inhibitors of PTKs. At least one exogenous cytosolic PTK, pp60^{c- src} , is also able to potentiate NMDA current. Because later application of PTK inhibitors can reverse the seemingly stable insulin-mediated potentiation of NMDA current, it appears that tyrosine residues responsible for potentiation are continually rephosphorylated by some long-term PTK activity that was induced via insulin treatment.     

Herpes simplex virus-1 up-regulates IL-15 gene expression in monocytic cells through the activation of protein tyrosine kinase and PKC zeta/lambda signaling pathways

IL-15 plays a seminal role in innate immunity through enhancing the cytotoxic function as well as cytokine production by NK and T cells. We have previously shown that exposure of PBMC as well as monocytic cells to different viruses results in immediate up-regulation of IL-15 gene expression and subsequent NK cell activation as an innate immune response of those cells to these viruses. However, no signaling pathway involved in this up-regulation has been identified. Here we show for the first time that HSV-1-induced up-regulation of IL-15 gene expression is independent of viral infectivity/replication. IL-15 gene is up-regulated by HSV-1 in human monocytes, but not in CD3+ T cells. HSV-1 induces the phosphorylation of protein tyrosine kinases (PTKs) and protein kinase C (PKC) for inducing IL-15 expression in monocytic cells. Inhibitors for PTKs reduced HSV-1-induced PTK activity, DNA binding activity of NF-kB as well as IL-15 gene expression. In contrast, an inhibitor for membrane-bound tyrosine kinases had no effect on these events. Experiments using PKC inhibitors revealed that phosphorylation of PKC zeta/lambda (PKC zeta/lambda), DNA binding activity of NF-kB and HSV-1-induced up-regulation of IL-15 were all decreased. Furthermore, we found that HSV-1-induced IL-15 up-regulation was also dependent on PTKs regulation of PKC phosphorylation. Thus, we conclude that IL-15 up-regulation in HSV-1-treated monocytic cells is dependent on the activity of both PTKs and PKC zeta/lambda.