Kinase Binding Domain Research Articles

C-Cbl Tyrosine Kinase-binding Domain Mutant G306E Abolishes the Interaction of c-Cbl with CD38 and Fails to Promote Retinoic Acid-induced Cell Differentiation and G0 Arrest

Retinoic acid (RA) causes HL-60 human myeloblastic leukemia cell myeloid differentiation that is dependent on MAPK signaling. The process is propelled by c-Cbl, which binds the CD38 receptor as part of a signaling complex generating MAPK signaling. Here we report that the capability of c-Cbl to do this is lost in the G306E tyrosine kinase-binding domain mutant. Unlike wild-type (WT) c-Cbl, the G306E mutant c-Cbl fails to propel RA-induced differentiation, and disrupts the normal association with CD38. The G306E mutant does, like WT c-Cbl, co-immunoprecipitate with Vav, Slp-76, and p38. But unlike WT c-Cbl, does not cause MAPK signaling. In contrast, the C381A Ring finger domain mutant functions like WT c-Cbl. It binds CD38 and is part of the same apparent c-Cbl/Slp-76/Vav/p38 signaling complex. The C381A mutant causes MAPK signaling and propels RA-induced differentiation. In addition to HL-60 cells and their WT or mutant c-Cbl stable transfectants, the c-Cbl/Vav/Slp-76 complex is also found in NB4 cells where c-Cbl was previously also found to bind CD38. The data are consistent with a model in which the G306E mutant c-Cbl forms a signaling complex that includes Slp-76, Vav, and p38; but does not drive MAPK signaling because it fails to bind the CD38 receptor. Without the G306E mutation the c-Cbl unites CD38 with the signaling complex and delivers a MAPK signal that drives RA-induced differentiation. The results demonstrate the importance of the Gly306 residue in the ability of c-Cbl to propel RA-induced differentiation.

C-Cbl and Cbl-b Act Redundantly to Protect Osteoclasts from Apoptosis and to Displace HDAC6 from β-Tubulin, Stabilizing Microtubules and Podosomes

c-Cbl and Cbl-b are highly conserved adaptor proteins that participate in integrin signaling, regulating cytoskeletal organization, motility, and bone resorption. Deletion of both c-Cbl and Cbl-b in mice leads to embryonic lethality, indicating that the two proteins perform essential redundant functions. To examine the redundant actions of c-Cbl and Cbl-b in osteoclasts, we depleted c-Cbl in Cbl-b(-/-) osteoclasts by using a short hairpin RNA. Depleting both Cbl proteins disrupted both the podosome belt and the microtubule network and decreased bone-resorbing activity. Stabilizing the microtubules with paclitaxel or inhibiting histone deacetylase 6 (HDAC6), which destabilizes microtubules by deacetylating beta-tubulin, protected both the microtubule network and the podosome belt. Examination of the mechanism involved demonstrated that the conserved four-helix bundle of c-Cbl's tyrosine kinase binding domain bound to beta-tubulin, and both c-Cbl and Cbl-b displaced HDAC6. In addition to the effects on microtubules and the podosome belt, depleting both Cbls significantly increased the levels of the proapoptotic protein Bim and apoptosis relative to the levels induced by eliminating either protein alone. Thus, both c-Cbl and Cbl-b promote bone resorption via the stabilization of microtubules, allowing the formation of the podosome belt in osteoclasts, and by promoting osteoclast survival.

Loss of Cbl-b Increases Osteoclast Bone-Resorbing Activity and Induces Osteopenia

Cbl proteins are multifunctional adaptor molecules that modulate cellular activity by targeting the ubiquitylating system, endocytic complexes, and other effectors to a wide variety of regulatory proteins, especially activated receptor and nonreceptor tyrosine kinases. Cbl and Cbl-b perform unique functions in various cells, in addition to redundant functions that are required for embryonic development. We previously showed that eliminating Cbl impaired osteoclast motility, which modestly delayed embryonic bone development. We now report that Cbl-b(-/-) mice are osteopenic, because of increased bone resorption with little compensating increase in bone formation. In vitro bone-resorbing activity and differentiation of osteoclast-like cells (OCLs) were increased, as were some RANKL-induced signaling events (activation of NF-kappaB and the mitogen-activated protein kinases extracellular signal-regulated kinase [ERK] and p38), suggesting that specific RANKL-activated mechanisms contribute to the increased rate of differentiation and bone-resorbing activity. Re-expressing Cbl-b in Cbl-b(-/-) OCLs normalized the increased bone-resorbing activity and overexpressing Cbl-b in wildtype OCLs inhibited bone resorption. Cbl was without effect in either wildtype or Cbl-b(-/-) OCLs. Functional tyrosine kinase binding (TKB) and RING finger domains were required for the rescue by Cbl-b. Thus, both Cbl and Cbl-b perform regulatory functions in osteoclasts that are unique to one or the other protein (i.e., functions that cannot be compensated by the other homolog). One of Cbl-b's unique functions in osteoclasts is to downregulate bone resorption.

Level of reactive oxygen species induced by p21WAF(1)/CIP(1) is critical for the determination of cell fate

p21(WAF(1)/)(CIP(1)) is a well-known cell cycle regulatory protein which is overexpressed in several cancer cell lines, and known to determine cell fate. We generated three recombinant adenovirus vectors that expressed either the full-length p21 (Ad-p21F), a p21 mutant with a deletion of the C-terminal proliferative cell nuclear antigen (PCNA) binding domain (Ad-p21N), or a p21 mutant with a deletion of the N-terminal cyclin-dependent kinase binding domain (Ad-p21C). We transfected these vectors into five cancer cell lines. Premature senescence was induced in all of the lines only following transfection with Ad-p21N and Ad-p21F. In addition, apoptosis was also induced in LoVo and HCT116 cells that harbored wild-type p53 and the reactive oxygen species (ROS) level was higher than in senescent cells. Finally, the induction of apoptosis was inhibited by using siRNA to downregulate p53. This observation implies that there is a feedback signaling loop involving p21/ROS/p53 in apoptotic responses. It appears to be, at least in part, driven by high levels of p21 protein. Next, we investigated the cell death effect of endogenous p21 protein on cell fate using sodium butyrate (NaB). Treatment with 1 mM NaB or 2 to 5 mM NaB induced senescence or apoptosis, respectively. The level of intracellular ROS in 5 mM NaB treated cells was 2-fold higher, compared with that in 1 mM NaB treated cells. We also demonstrated that DNA damage response signals including ataxia telangiectasia mutated, gammaH2AX, and p38 MAPK were involved in NaB-induced cell death. The magnitude of intracellular ROS levels in response to p21 elicited either senescence or apoptosis in the cancer cell lines.

The Ile646Val (2073A>G) Polymorphism in the Kinase-Binding Domain of A-Kinase Anchoring Protein 10 and the Risk of Colorectal Cancer

Objective: The purpose of this study is to investigate whether the Ile646Val (2073A>G) polymorphism in the kinase-binding domain of A-kinase anchoring protein 10 (AKAP10) is related to the risk of colorectal cancer (CRC), clinicopathological variables and the environmental factors for the development of CRC. Methods: Applying TaqMan allelic discrimination, we investigated AKAP10 Ile646Val (2073A>G) polymorphism in 288 Chinese CRC patients and 281 healthy controls. Results: Logistic regression analysis revealed a significant association of AKAP10 Ile646Val (2073A>G) polymorphism with increased CRC risk (adjusted OR = 1.44, 95% CI 1.01–2.07, p = 0.02). Stratification analysis showed that the increased risk associated with the variant genotypes (GG+AG) was more evident in male subjects (adjusted OR = 1.48, 95% CI 0.94–2.34, p = 0.03). Compared with the AA genotype, the adjusted OR for the variant genotypes was 1.81 (95% CI 1.08–3.05, p = 0.01) among young subjects (age <57 years). Among individuals who did not smoke or who smoked lightly, there was a significantly increased risk with the variant genotypes (adjusted OR = 1.66, 95% CI 1.08–2.56, p = 0.02). We did not observe a relationship between the AKAP10 polymorphism and other clinicopathological and environmental factors. Conclusions: Our data suggested that the AKAP10 2073A>G variation is associated with an increased risk of CRC in the Chinese population.

The Role of Quaternary Structure in the Signaling Mechanisms of PAS Sensor Domains

The modular nature of proteins containing PAS (Per-ARNT-Sim) or other sensor domains enables signaling networks to be diverse and poses an interesting question: how can sensor domains with largely conserved tertiary structures regulate effector domains with very diverse structures and functions? We address this question by examining signal processing by the PAS sensor domain, which can regulate the activity of covalently linked effector domains such as a kinase, phosphodiesterase or DNA binding domains. In many cases oligomerization of sensor proteins is essential for signal transduction. We present the structure of a heme-PAS domain dimer from Bradyrhizobium japonicum (bjFixLH) in a new space group (P1) and at higher resolutions (1.5-1.8 A) than those previously obtained. Interestingly, bjFixLH can form two different dimers in the same crystallization solution, where the monomers in one dimer are rotated ∼175° relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. Comparison of PAS domain dimers using screw rotation analysis reveals that PAS monomers adopt a discrete range of monomer orientations. Similar to the light-sensitive PAS domain YtvA-LOV from Bacillus subtilis, bjFixLH undergoes signal-induced quaternary structural changes where monomers rotate ∼2° relative to each other. Signal-induced quaternary structural changes accommodate the ability of PAS sensor domains to regulate a wide variety of effector domains since PAS and effector domains would not be required to interact with each other in a structure-specific manner. Our results will guide the rational design of novel PAS signaling proteins.

C-Jun N-terminal kinase binding domain–dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions.This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase–binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1–ERK–Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway—(MEK1/ERK) as a consequence of the death pathway—(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

Enzymatic Activity and Substrate Specificity of Mitogen-activated Protein Kinase p38α in Different Phosphorylation States

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38alpha, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38alpha phosphorylated at both Thr-180 and Tyr-182 was 10-20-fold more active than p38alpha phosphorylated at Thr-180 only, whereas p38alpha phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Calpha are all highly specific for the dephosphorylation of p38alpha, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38alpha; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k(cat)/K(m) for the dephosphorylation of p38alpha. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38alpha and to the substrate specificity for various phosphatases.

Structural basis for a novel intrapeptidyl H-bond and reverse binding of c-Cbl-TKB domain substrates

The c-Cbl tyrosine kinase binding domain (Cbl-TKB), essentially an 'embedded' SH2 domain, has a critical role in targeting proteins for ubiquitination. To address how this domain can bind to disparate recognition mofits and to determine whether this results in variations in substrate-binding affinity, we compared crystal structures of the Cbl-TKB domain complexed with phosphorylated peptides of Sprouty2, Sprouty4, epidermal growth factor receptor, Syk, and c-Met receptors and validated the binding with point-mutational analyses using full-length proteins. An obligatory, intrapeptidyl H-bond between the phosphotyrosine and the conserved asparagine or adjacent arginine is essential for binding and orients the peptide into a positively charged pocket on c-Cbl. Surprisingly, c-Met bound to Cbl in the reverse direction, which is unprecedented for SH2 domain binding. The necessity of this intrapeptidyl H-bond was confirmed with isothermal titration calorimetry experiments that also showed Sprouty2 to have the highest binding affinity to c-Cbl; this may enable the selective sequestration of c-Cbl from other target proteins.

A Novel Role of Sprouty 2 in Regulating Cellular Apoptosis

Sprouty (SPRY) proteins modulate receptor-tyrosine kinase signaling and, thereby, regulate cell migration and proliferation. Here, we have examined the role of endogenous human SPRY2 (hSPRY2) in the regulation of cellular apoptosis. Small inhibitory RNA-mediated silencing of hSPRY2 abolished the anti-apoptotic action of serum in adrenal cortex adenocarcinoma (SW13) cells. Silencing of hSPRY2 decreased serum- or epidermal growth factor (EGF)-elicited activation of AKT and ERK1/2 and reduced the levels of EGF receptor. Silencing of hSPRY2 also inhibited serum-induced activation of p90RSK and decreased phosphorylation of pro-apoptotic protein BAD (BCL2-antagonist of cell death) by p90RSK. Inhibiting both the ERK1/2 and AKT pathways abolished the ability of serum to protect against apoptosis, mimicking the effects of silencing hSPRY2. Serum transactivated the EGF receptor (EGFR), and inhibition of the EGFR by a neutralizing antibody attenuated the anti-apoptotic actions of serum. Consistent with the role of EGFR and perhaps other growth factor receptors in the anti-apoptotic actions of serum, the tyrosine kinase binding domain of c-Cbl (Cbl-TKB) protected against down-regulation of the growth factor receptors such as EGFR and preserved the anti-apoptotic actions of serum when hSpry2 was silenced. Additionally, silencing of Spry2 in c-Cbl null cells did not alter the ability of serum to promote cell survival. Moreover, reintroduction of wild type hSPRY2, but not its mutants that do not bind c-Cbl or CIN85 into SW13 cells after endogenous hSPRY2 had been silenced, restored the anti-apoptotic actions of serum. Overall, we conclude that endogenous hSPRY2-mediated regulation of apoptosis requires c-Cbl and is manifested by the ability of hSPRY2 to sequester c-Cbl and thereby augment signaling via growth factor receptors.

Ligand Binding Induces Cbl‐Dependent EphB1 Receptor Degradation Through the Lysosomal Pathway

Eph receptor tyrosine kinases play a critical role in embryonic patterning and angiogenesis. In the adult, they are involved in carcinogenesis and pathological neovascularization. However, the mechanisms underlying their role in tumor formation and metastasis remain to be defined. Here, we demonstrated that stimulation of EphB1 with ephrinB1/Fc led to a marked downregulation of EphB1 protein, a process blocked by the lysosomal inhibitor bafilomycin. Following ephrinB1 stimulation, the ubiquitin ligase Cbl was recruited by EphB1 and then phosphorylated. Both Cbl phosphorylation and EphB1 ubiquitination were blocked by the Src inhibitor PP2. Overexpression of wild-type Cbl, but not of 70Z mutant lacking ligase activity, enhanced EphB1 ubiquitination and degradation. This negative regulation required the tyrosine kinase activity of EphB1 as kinase-dead EphB1-K652R was resistant to Cbl. Glutathione S-transferase binding experiments showed that Cbl bound to EphB1 through its tyrosine kinase-binding domain. In aggregate, we demonstrated that Cbl induces the ubiquitination and lysosomal degradation of activated EphB1, a process requiring EphB1 and Src kinase activity. To our knowledge, this is the first study dissecting the molecular mechanisms leading to EphB1 downregulation, thus paving the way to new means of modulating their angiogenic and tumorigenic properties.

Hsp70 suppresses apoptosis in sympathetic neurones by preventing the activation of c‐Jun

The anti-apoptotic effects of heat-shock protein (Hsp70) were assessed in SCG neurones following nerve growth factor (NGF) withdrawal. The results showed that the virally mediated expression of Hsp70 mirrored the effects of the c-Jun-N-terminal kinase (JNK) binding domain (JBD) of JNK interacting protein (an inhibitor of JNK and c-Jun activation) and suppressed the phosphorylation of c-Jun. Preventing c-Jun transcriptional activity subsequently led to reduced cytochrome c release and prevented caspase activation as indicated by a decrease in poly (ADP-ribose) polymerase-1 (PARP) cleavage. Together, these results show that Hsp70 is a highly effective inhibitor of apoptosis in sympathetic neurones and that it mediates this effect primarily by suppressing c-Jun transcriptional signalling.

Degradation of HER2 by Cbl-Based Chimeric Ubiquitin Ligases

Targeting disease-causing proteins for ubiquitination and degradation by chimeric molecules represents a promising alternative therapeutic strategy in cancer. Here, several Cbl-based chimeric ubiquitin ligases were recombined to achieve effective down-regulation of HER2. These chimeric molecules consisted of the Cbl NH(2)-terminal tyrosine kinase binding domain, linker, and RING domain, with the Src homology 2 domain replaced with that from growth factor receptor binding protein 2 (Grb2), Grb7, p85, or Src. The chimeric proteins not only interacted with HER2 but also enhanced the down-regulation of endogenous overexpressed HER2. After the chimeric proteins were introduced into HER2-overexpressing breast cancer SK-BR-3 cells or ovarian cancer SK-OV-3 cells, they effectively promoted HER2 ubiquitination and degradation in a RING finger domain-dependent manner. Consequently, expression of these chimeric molecules led to an inhibition of colony formation, increased the proportion of cells in the G(1) cycle, and suppressed tumorigenicity. Collectively, our findings suggest that the Cbl-based chimeric ubiquitin ligases designed in the present study may represent a novel approach for the targeted therapy of HER2-overexpressing cancers.

Structural Basis for UBA-mediated Dimerization of c-Cbl Ubiquitin Ligase

Ligand-induced down-regulation by the ubiquitin-protein ligases, c-Cbl and Cbl-b, controls signaling downstream from many receptor-tyrosine kinases (RTK). Cbl proteins bind to phosphotyrosine residues on activated RTKs to affect ligand-dependent ubiquitylation of these receptors targeting them for degradation in the lysosome. Both c-Cbl and Cbl-b contain a ubiquitin-associated (UBA) domain, which is important for Cbl dimerization and tyrosine phosphorylation; however, the mechanism of UBA-mediated dimerization and its requirement for Cbl biological activity is unclear. Here, we report the crystal structure of the UBA domain of c-Cbl refined to 2.1-A resolution. The structure reveals the protein is a symmetric dimer tightly packed along a large hydrophobic surface formed by helices 2 and 3. NMR chemical shift mapping reveals heterodimerization can occur with the related Cbl-b UBA domain via the same surface employed for homodimerization. Disruption of c-Cbl dimerization by site-directed mutagenesis impairs c-Cbl phosphorylation following activation of the Met/hepatocyte growth factor RTK and c-Cbl-dependent ubiquitination of Met. This provides direct evidence for a role of Cbl dimerization in terminating signaling following activation of RTKs.

Impact of NS5A Sequences of Hepatitis C Virus Genotype 1a on Early Viral Kinetics during Treatment with Peginterferon‐α2a plus Ribavirin

BACKGROUND. Hepatitis C virus (HCV) genotype 1 is the most prevalent genotype in Western countries, and treatment with pegylated interferon (pegIFN) plus ribavirin fails in 50%-60% of patients. Genetic variability within the NS5A dsRNA-dependent protein kinase binding domain (PKRbd) of HCV-1b has been associated with responsiveness to IFN- alpha . Little is known about NS5A sequences of HCV-1a. We investigated whether genetic variability of HCV-1a NS5A correlates with the early HCV kinetics during treatment. Twenty-four treatment-naive, HCV-1a-infected patients were treated with standard doses of pegIFN- alpha 2a plus ribavirin. HCV viremia was quantitated at days 0, 1, 2, and 3 and weeks 1, 2, 4, 8, and 12 of treatment. According to HCV kinetics, patients were classified as early rapid responders, early moderate responders, or early slow responders. The full-length HCV NS5A was sequenced at baseline and at week 1. At baseline, variability of the NS5A C terminus (concentrated in the PKRbd) is associated with interferon efficacy but not with the second phase of the early viral decline that has been associated with a sustained virologic response. Comparisons between baseline and week-1 full-length sequences did not show significant increases in mutations. Genetic variability of HCV-1a NS5A does not predict responsiveness to IFN- alpha . Differences in early kinetics during combination therapy are not due to selection of IFN-resistant HCV strains.

A DOUBLETIME kinase binding domain on the Drosophila PERIOD protein is essential for its hyperphosphorylation, transcriptional repression, and circadian clock function.

A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins from hypo- to hyperphosphorylated species, events that are highly dependent on casein kinase 1 epsilon (termed DOUBLETIME [DBT] in Drosophila melanogaster) and necessary for normal clock progression. Drosophila PER (dPER) functions in the negative limb of the clockworks by presumably binding to the transcription factor CLOCK (CLK) and inhibiting its transactivation activity. Here, we identify a small region on dPER that is conserved with mammalian PERs and contains the major in vivo DBT binding domain, termed dPDBD (for dPER DBT binding domain). This domain is required for the manifestation of molecular and behavioral rhythms in vivo. In the absence of the dPDBD, the dPER protein is present at constant high levels throughout a daily cycle, undergoes little phosphorylation, and is severely impaired in its ability to function as a transcriptional repressor. Our findings indicate that the binding of dPER to CLK is not sufficient for transcriptional inhibition, implicating a more indirect mode of action whereby dPER acts as a molecular bridge to "deliver" DBT and/or other factors that directly repress CLK-dependent gene expression.

Alteration of the Protein Kinase Binding Domain Enhances Function of the Saccharomyces cerevisiae Molecular Chaperone Cdc37

Cdc37 is a molecular chaperone that has a general function in the biogenesis of protein kinases. We identified mutations within the putative "protein kinase binding domain" of Cdc37 that alleviate the conditional growth defect of a strain containing a temperature-sensitive allele, tpk2(Ts), of the cyclic AMP-dependent protein kinase (PKA). These dominant mutations alleviate the temperature-sensitive growth defect by elevating PKA activity, as judged by their effects on PKA-regulated processes, localization and phosphorylation of the PKA effector Msn2, as well as in vitro PKA activity. Although the tpk2(Ts) growth defect is also alleviated by Cdc37 overproduction, the CDC37 dominant mutants contain wild-type Cdc37 protein levels. In addition, Saccharomyces cerevisiae Ste11 protein kinase has an elevated physical interaction with the altered Cdc37 protein. These results implicate specific amino-terminal residues in the interaction between Cdc37 and client protein kinases and provide further genetic and biochemical support for a model in which Cdc37 functions as a molecular chaperone for protein kinases.

Expression and translocation of fluorescent-tagged p21-activated kinase-binding domain and PH domain of protein kinase B during murine neutrophil chemotaxis

Neutrophils are key cells of the innate immune system; they are terminally differentiated and therefore difficult to genetically manipulate and study in vitro. In the present study, we describe a protocol to transiently express two fluorescent markers, the PH domain of protein kinase B fused to red fluorescent protein and the p21-activated kinase-binding domain fused to a yellow fluorescent protein, in primary neutrophils. Using this approach, we are able to achieve a transfection efficiency of approximately 30%. The expression of the transfected probes occurred within 2 h and allowed for real-time monitoring of intermediates in key neutrophil activation pathways at the leading edge of migrating cells. We describe here a transfection protocol for primary neutrophils, which preserves fMLP-mediated cell polarization and cytoskeleton reorganization with simultaneous accumulation of PI-3K products and active Rac at the leading edge. The visualization and analysis of transfected fluorescent markers in primary neutrophils are a powerful technique to monitor chemotaxis signaling pathways in real time.

Crystal structure of the MAP kinase binding domain and the catalytic domain of human MKP5.

MAP kinase phosphatases (MKPs) have crucial roles in regulating the signaling activity of MAP kinases and are potential targets for drug discovery against human diseases. These enzymes contain a catalytic domain (CD) as well as a binding domain (BD) that help recognize the target MAP kinase. We report here the crystal structures at up to 2.2 A resolution of the BD and CD of human MKP5 and compare them to the known structures from other MKPs. Dramatic structural differences are observed between the BD of MKP5 and that of MKP3 determined previously by NMR. In particular, the cluster of positively charged residues that is important for MAP kinase binding is located in completely different positions in the two structures, with a distance of 25 A between them. Moreover, this cluster is alpha-helical in MKP5, while it forms a loop followed by a beta-strand in MKP3. These large structural differences could be associated with the distinct substrate preferences of these phosphatases, but further studies are needed to confirm this. The CD of MKP5 is observed in an active conformation, and two loops in the active site have backbone shifts of up to 5 A relative to the inactive CDs from other MKPs.

Sensitization of vascular smooth muscle cell to TNF-α-mediated death in the presence of palmitate

Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-α-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokine did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-α-mediated nuclear factor kappa B (NF-κB) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-κB subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-κB predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-α-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in deleterious vascular consequences of such patients with elevated levels of FFAs as diabetics and obese individuals via the triggering of receptor-mediated death pathways of VSMC.