Activation Loop Site Research Articles

Investigation of the Signaling Mechanisms of the Mu‐Delta Opioid Receptor Heterodimer

It has been hypothesized that the mu‐ and delta‐opioid receptors (MOR and DOR) can form a heterodimer (MDOR) and that the resulting heterodimer has a distinct pharmacology. Limited evidence from the literature suggests that MDOR‐selective antagonists could be a novel therapy to enhance opioid efficacy while decreasing side effects. However, due to the lack of selective tools, it is very difficult to study this complex system. We thus recently developed D24M, a novel, first‐in‐class selective MDOR antagonist. When D24M was combined with oxymorphone in vivo, we found potentiation of antinociception and reduction of withdrawal symptoms, validating the hypothesis of the MDOR as an anti‐opioid negative feedback loop and that MDOR antagonists could be novel therapeutics. These striking results prompted us to investigate the underlying signaling mechanisms of the MDOR. First, we treated male and female CD‐1 mice with oxymorphone and/or D24M, analyzed the brainstem using quantitative phospho‐proteomic analysis. Out of the thousands of hits, we found dozens of potential targets that became phosphorylated or dephosphorylated upon exposure to both oxymorphone and D24M. Based on this phospho‐proteomic database, we selected the signaling kinases CaMKII and Src to further investigate in vivo. We ran Western blot and tail‐flick anti‐nociception experiments in male and female CD‐1 mice in the presence or absence of specific kinase inhibitors (KN93 and Src I1) combined with oxymorphone and/or D24M. We found that both Src and CaMKII inhibitors blocked the enhanced anti‐nociception caused by D24M, while not impacting baseline opioid response. These results suggest that Src and CaMKII are both repressed by the MDOR to repress opioid anti‐nociception, which is reversed by D24M treatment. However, Western blot analysis did not show phosphorylation differences in the canonical activation loop sites of either kinase, suggesting that alternate phosphorylation sites identified in our proteomic analysis may be responsible for the signaling effects of these kinases downstream of the MDOR. Our results have thus identified 2 key nodes in the MDOR signal transduction cascade by which the MDOR blocks opioid anti‐nociception, and begun to explore their molecular signaling mechanisms. Future work will expand on the signaling cascades of the MDOR, as well as further establish MDOR antagonists as novel opioid therapeutics.Support or Funding InformationThese studies were funded by NIH R21DA044509 and UG3DA047717 to JMS. The authors have no relevant conflicts of interest to declare.

Functional characterization of Mitogen-Activated Protein Kinase Kinase (MAPKK) gene in Halophytic Salicornia europaea against salt stress

Mitogen-activated protein kinase (MAPK) cascade is conserved in eukaryotic organisms and plays a crucial role in signal transduction pathways, related to biotic and abiotic stress responses. The current study was aimed to target at cloning, structural and functional characterization of 1023bp SeMAPKK gene from Salicornia europaea. A SeMAPKK polypeptide chain of 340aa has eleven sub-domains, and S/TXXXXXS/T residues are conserved in the activation loop site between VII-VIII sub-domains. We identified TVY and MEY motifs at N-terminus and LKY, CAY and SKY motifs at C-terminus. Phylogenetic relationship revealed that SeMAPKK belongs to group D. Highest transcript signals were recorded in 0.75 M salt level stress, and down-regulation was observed in 1.0 M salt concentration. Overexpression of SeMAPKK gene in Arabidopsis conferred salt tolerance. Results of different salt tolerance assays revealed that transgenic plants were more tolerant and exhibited better growth than non-transgenic plants under salt stress.

Insulin promotes proliferation and fibrosing responses in activated pancreatic stellate cells.

Epidemiological studies support strong links between obesity, diabetes, and pancreatic disorders including pancreatitis and pancreatic adenocarcinoma (PDAC). Type 2 diabetes (T2DM) is associated with insulin resistance, hyperglycemia, and hyperinsulinemia, the latter due to increased insulin secretion by pancreatic beta-cells. We reported that high-fat diet-induced PDAC progression in mice is associated with hyperglycemia, hyperinsulinemia, and activation of pancreatic stellate cells (PaSC). We investigated here the effects of high concentrations of insulin and glucose on mouse and human PaSC growth and fibrosing responses. We found that compared with normal, pancreata from T2DM patients displayed extensive collagen deposition and activated PaSC in islet and peri-islet exocrine pancreas. Mice fed a high-fat diet for up to 12 mo similarly displayed increasing peri-islet fibrosis compared with mice fed control diet. Both quiescent and activated PaSC coexpress insulin (IR; mainly A type) and IGF (IGF-1R) receptors, and both insulin and glucose modulate receptor expression. In cultured PaSC, insulin induced rapid tyrosine autophosphorylation of IR/IGF-1R at specific kinase domain activation loop sites, activated Akt/mTOR/p70S6K signaling, and inactivated FoxO1, a transcription factor that restrains cell growth. Insulin did not promote activation of quiescent PaSC in either 5 mM or 25 mM glucose containing media. However, in activated PaSC, insulin enhanced cell proliferation and augmented production of extracellular matrix proteins, and these effects were abolished by specific inhibition of mTORC1 and mTORC2. In conclusion, our data support the concept that increased local glucose and insulin concentrations associated with obesity and T2DM promote PaSC growth and fibrosing responses.

Gene structure, molecular characterization and transcriptional expression of two p38 isoforms (MAPK11 and MAPK14) from rock bream (Oplegnathus fasciatus).

The p38 kinases are one of the four subgroups of mitogen-activated protein kinase (MAPK) superfamily which are involved in the innate immunity. The p38 subfamily that includes four members namely p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), regulates the activation of several transcription factors. In this study, a p38β (OfMAPK11) homolog and a p38α (OfMAPK14) homolog of Oplegnathus fasciatus were identified at genomic level. Results clearly showed that both MAPK11 and MAPK14 are well-conserved at both genomic structural- and amino acid (aa)-levels. Genomic sequences of OfMAPK11 (∼ 15.6 kb) and OfMAPK14 (∼ 13.4 kb) had 12 exons. A comparison of exon-intron structural arrangement of these genes from different vertebrate lineages indicated that all the exon lengths are highly conserved, except their terminal exons. Full-length cDNAs of OfMAPK11 (3957 bp) and OfMAPK14 (2504 bp) encoded corresponding proteins of 361 aa and 360 aa, respectively. Both OfMAPK proteins harbored a Ser/Thr protein kinases catalytic domain (S_TKc domain) which includes an activation loop with a dual phosphorylation site (TGY motif) and several specific-binding sites for ATP and substrates. Molecular modeling of the activation loop and substrate binding sites of rock bream MAPKs revealed the conservation of crucial residues and their orientation in 3D space. Transcripts of OfMAPKs were ubiquitously detected in eleven tissues examined, however at different levels. The modulation of OfMAPKs' transcription upon pathogen-associated molecular patterns (PAMPs: flagellin, lipopolysaccharide and poly I:C) and pathogens (Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus) was investigated. Among the seven examined tissues, the flagellin-challenge upregulated the mRNA level of both OfMAPKs in the head kidney. Meanwhile, modulation of OfMAPK mRNA expression in the liver upon other immune-challenges varied in a time-dependent manner. Collectively, these results suggest that OfMAPKs are true members of p38 subfamily, which might be induced by different immune stimuli.

Novel Phosphotidylinositol 4,5-Bisphosphate Binding Sites on Focal Adhesion Kinase.

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is ubiquitously expressed, recruited to focal adhesions, and engages in a variety of cellular signaling pathways. Diverse cellular responses, such as cell migration, proliferation, and survival, are regulated by FAK. Prior to activation, FAK adopts an autoinhibited conformation in which the FERM domain binds the kinase domain, blocking access to the activation loop and substrate binding site. Activation of FAK occurs through conformational change, and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2) are known to facilitate this process. PIP2 binding alters the autoinhibited conformation of the FERM and kinase domains and subsequently exposes the activation loop to phosphorylation. However, the detailed molecular mechanism of PIP2 binding and its role in FAK activation remain unclear. In this study, we conducted coarse-grained molecular dynamics simulations to investigate the binding of FAK to PIP2. Our simulations identified novel areas of basic residues in the kinase domain of FAK that potentially undergo transient binding to PIP2 through electrostatic attractions. Our investigation provides a molecular picture of PIP2-initiated FAK activation and introduces promising new pathways for future studies of FAK regulation.

Crystal Structure of a Human IκB Kinase β Asymmetric Dimer

Phosphorylation of inhibitor of nuclear transcription factor κB (IκB) by IκB kinase (IKK) triggers the degradation of IκB and migration of cytoplasmic κB to the nucleus where it promotes the transcription of its target genes. Activation of IKK is achieved by phosphorylation of its main subunit, IKKβ, at the activation loop sites. Here, we report the 2.8 Å resolution crystal structure of human IKKβ (hIKKβ), which is partially phosphorylated and bound to the staurosporine analog K252a. The hIKKβ protomer adopts a trimodular structure that closely resembles that from Xenopus laevis (xIKKβ): an N-terminal kinase domain (KD), a central ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (SDD). Although hIKKβ and xIKKβ utilize a similar dimerization mode, their overall geometries are distinct. In contrast to the structure resembling closed shears reported previously for xIKKβ, hIKKβ exists as an open asymmetric dimer in which the two KDs are further apart, with one in an active and the other in an inactive conformation. Dimer interactions are limited to the C-terminal six-helix bundle that acts as a hinge between the two subunits. The observed domain movements in the structures of IKKβ may represent trans-phosphorylation steps that accompany IKKβ activation.

The activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases in Saccharomyces cerevisiae

PDK1 (phosphoinositide-dependent protein kinase 1) phosphorylates and activates PKA (cAMP-dependent protein kinase) in vitro. Docking of the HM (hydrophobic motif) in the C-terminal tail of the PKA catalytic subunits on to the PIF (PDK1-interacting fragment) pocket of PDK1 is a critical step in this activation process. However, PDK1 regulation of PKA in vivo remains controversial. Saccharomyces cerevisiae contains three PKA catalytic subunits, TPK1, TPK2 and TPK3. We demonstrate that Pkh [PKB (protein kinase B)-activating kinase homologue] protein kinases phosphorylate the activation loop of each Tpk in vivo with various efficiencies. Pkh inactivation reduces the interaction of each catalytic subunit with the regulatory subunit Bcy1 without affecting the specific kinase activity of PKA. Comparative analysis of the in vitro interaction and phosphorylation of Tpks by Pkh1 shows that Tpk1 and Tpk2 interact with Pkh1 through an HM-PIF pocket interaction. Unlike Tpk1, mutagenesis of the activation loop site in Tpk2 does not abolish in vitro phosphorylation, suggesting that Tpk2 contains other, as yet uncharacterized, Pkh1 target sites. Tpk3 is poorly phosphorylated on its activation loop site, and this is due to the weak interaction of Tpk3 with Pkh1 because of the atypical HM found in Tpk3. In conclusion, the results of the present study show that Pkh protein kinases contribute to the divergent regulation of the Tpk catalytic subunits.

Differential PKC-dependent and -independent PKD activation by G protein α subunits of the Gq family: Selective stimulation of PKD Ser748 autophosphorylation by Gαq

Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gαq-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family Gα and stimulated with aluminum fluoride (AlF4−). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser744, a prominent and rapid PKC transphosphorylation site, and Ser748, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4− potently induced PKD activation and Ser744 and Ser748 phosphorylation, in the presence of cotransfected Gαq, Gα11, Gα14 or Gα15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked Gα11-, Gα14-, and Gα15-mediated enhancement of Ser748 phosphorylation induced by AlF4−, and largely abolished Ser744 phosphorylation. In contrast, Ser748 phosphorylation was almost completely intact, and Ser744 phosphorylation was significantly activated in cells cotransfected with Gαq. Importantly, the differential Ser748 phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gαq but not Gα11. Taken together, our results suggest that Gαq, but not the closely related Gα11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser748.

Cocaine facilitates PKC maturation by upregulating its phosphorylation at the activation loop in rat striatal neurons in vivo

Newly synthesized protein kinase C (PKC) undergoes a series of phosphorylation to render a mature form of the enzyme. It is this mature PKC that possesses the catalytic competence to respond to second messengers for activation and downstream signaling. The first and rate-limiting phosphorylation occurs at a threonine residue in the activation loop (AL), which triggers the rest maturation processing of PKC and regulates PKC activity in response to cellular stimulation. Given the fact that PKC is enriched in striatal neurons, we investigated the regulation of PKC phosphorylation at the AL site in the rat striatum by the psychostimulant cocaine in vivo. We found that PKC was phosphorylated at the AL site at a moderate level in the normal rat brain. Acute systemic injection of cocaine increased the PKC-AL phosphorylation in the two striatal structures (caudate putamen and nucleus accumbens). Cocaine also elevated the PKC-AL phosphorylation in the medial prefrontal cortex. The cocaine-stimulated PKC phosphorylation in the striatum is rapid and transient. A reliable increase in PKC phosphorylation was seen 7min after drug injection, which declined to the normal level by 1h. This kinetics corresponds to that seen for another striatum-enriched protein kinase, mitogen-activated protein kinase/extracellular signal-regulated kinase, in response to cocaine. This study suggests a new model for exploring the impact of cocaine on protein kinases in striatal neurons. By modifying PKC phosphorylation at the AL site, cocaine is believed to possess the ability to alter the maturation processing of the kinase in striatal neurons in vivo.

Disruption of the Interface between the Pleckstrin Homology (PH) and Kinase Domains of Akt Protein Is Sufficient for Hydrophobic Motif Site Phosphorylation in the Absence of mTORC2

The pro-survival kinase Akt requires phosphorylation at two conserved residues, the activation loop site (Thr-308) and the hydrophobic motif site (Ser-473), for maximal activation. Previous reports indicate that mTORC2 is necessary for phosphorylation of the hydrophobic motif and that this site is not phosphorylated in cells lacking components of the mTORC2 complex, such as Sin1. Here we show that Akt can be phosphorylated at the hydrophobic motif site (Ser-473) in the absence of mTORC2. First, increasing the levels of PIP(3) in Sin1(-/-) MEFs by (i) expression of a constitutively active PI3K or (ii) relief of a negative feedback loop on PI3K by prolonged inhibition of mTORC1 or S6K is sufficient to rescue hydrophobic motif phosphorylation of Akt. The resulting accumulation of PIP(3) at the plasma membrane results in Ser-473 phosphorylation. Second, constructs of Akt in which the PH domain is constitutively disengaged from the kinase domain are phosphorylated at the hydrophobic motif site in Sin1(-/-) MEFs; both myristoylated-Akt and Akt lacking the PH domain are phosphorylated at Ser-473. Thus, disruption of the interface between the PH and kinase domains of Akt bypasses the requirement for mTORC2. In summary, these data support a model in which Akt can be phosphorylated at Ser-473 and activated in the absence of mTORC2 by mechanisms that depend on removal of the PH domain from the kinase domain.

TBK1 Directly Engages Akt/PKB Survival Signaling to Support Oncogenic Transformation

The innate immune-signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here, we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low-nanomolar TBK1 inhibitor, indicates that this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling.

The Third Conformation of p38α MAP Kinase Observed in Phosphorylated p38α and in Solution

MAPKs engage substrates, MAP2Ks, and phosphatases via a docking groove in the C-terminal domain of the kinase. Prior crystallographic studies on the unphosphorylated MAPKs p38α and ERK2 defined the docking groove and revealed long-range conformational changes affecting the activation loop and active site of the kinase induced by peptide. Solution NMR data presented here for unphosphorylated p38α with a MEK3b-derived peptide (p38α/pepMEK3b) validate these findings. Crystallograhic data from doubly phosphorylated active p38α (p38α/T∗GY∗/pepMEK3b) reveal a structure similar to unphosphorylated p38α/MEK3b, and distinct from phosphorylated p38γ (p38γ/T∗GY∗) and ERK2 (ERK2/T∗EY∗). The structure supports the idea that MAP kinases adopt three distinct conformations: unphosphorylated, phosphorylated, and a docking peptide-induced form.

Protein kinase D mediates synergistic expression of COX-2 induced by TNF-α and bradykinin in human colonic myofibroblasts

Myofibroblasts have recently been identified as major mediators of tumor necrosis factor-alpha (TNF-alpha)-associated colitis, but the precise mechanism(s) involved remains incompletely understood. In particular, the possibility that TNF-alpha signaling cross talks with other proinflammatory mediators, including bradykinin (BK), has not been examined in these cells. Here we show that treatment of 18Co cells, a model of human colonic myofibroblasts, with BK and TNF-alpha induced striking synergistic COX-2 protein expression that was paralleled by increases in the levels of transcripts encoding COX-2 and microsomal prostaglandin E synthase 1 (mPGES-1) and by the production of PGE(2). COX-2 expression in 18Co cells treated with BK and TNF-alpha was prevented by the B(2) BK receptor antagonist HOE-140, the preferential protein kinase C (PKC) inhibitors Ro31-8220 and GF-109203X, and Gö-6976, an inhibitor of conventional PKCs and protein kinase D (PKD). In a parallel fashion, TNF-alpha, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation induced by BK, as measured by PKD phosphorylation at its activation loop (Ser(744)) and autophosphorylation site (Ser(916)). BK-induced PKD activation was also inhibited by HOE-140, Ro31-8220, and Gö-6976. Transfection of 18Co cells with small interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein in response to BK and TNF-alpha, demonstrating, for the first time, a critical role of PKD in the pathways leading to synergistic expression of COX-2. Our results imply that cross talk between TNF-alpha and BK amplifies a PKD phosphorylation cascade that mediates synergistic COX-2 expression in colonic myofibroblasts. It is plausible that PKD increases COX-2 expression in colonic myofibroblasts to promote an inflammatory microenvironment that supports tumor growth.

Regulation of protein kinase Cδ downregulation by protein kinase Cε and mammalian target of rapamycin complex 2

Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKCδ by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKCδ downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKCδ at the activation loop (Thr505), turn motif (Ser643), hydrophobic motif (Ser662) and Tyr-311 sites to a greater extent in HeLa/CP cells compared to HeLa cells. Prolonged treatment with PDBu led to downregulation of PKCδ in HeLa but not in HeLa/CP cells. The PKC inhibitor Gö 6983 inhibited PDBu-induced downregulation of PKCδ, decreased Thr505 phosphorylation and increased PKCδ tyrosine phosphorylation at Tyr-311 site. However, knockdown of c-Abl, c-Src, Fyn and Lyn had little effect on PKCδ downregulation and Tyr311 phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor Ly294002 and mTOR inhibitor rapamycin restored the ability of PDBu to downregulate PKCδ in HeLa/CP cells. Knockdown of mTOR and rictor but not raptor facilitated PKCδ downregulation. Depletion of PKCε also enhanced PKCδ downregulation by PDBu. These results suggest that downregulation of PKCδ is regulated by PKCε and mammalian target of rapamycin complex 2 (mTORC2).

Protein kinase D1 mediates NF-kappaB activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells.

The transcription factor NF-kappaB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.

TCR-induced downregulation of protein tyrosine phosphatase PEST augments secondary T cell responses

We report that the protein tyrosine phosphatase PTP-PEST is expressed in resting human and mouse CD4 + and CD8 + T cells, but not in Jurkat T leukemia cells, and that PTP-PEST protein, but not mRNA, was dramatically downregulated in CD4 + and CD8 + primary human T cells upon T cell activation. This was also true in mouse CD4 + T cells, but less striking in mouse CD8 + T cells. PTP-PEST reintroduced into Jurkat at levels similar to those in primary human T cells, was a potent inhibitor of TCR-induced transactivation of reporter genes driven by NFAT/AP-1 and NF-κB elements and by the entire IL-2 gene promoter. Introduction of PTP-PEST into previously activated primary human T cells also reduced subsequent IL-2 production by these cells in response to TCR and CD28 stimulation. The inhibitory effect of PTP-PEST was associated with dephosphorylation the Lck kinase at its activation loop site (Y394), reduced early TCR-induced tyrosine phosphorylation, reduced ZAP-70 phosphorylation and inhibition of MAP kinase activation. We propose that PTP-PEST tempers T cell activation by dephosphorylating TCR-proximal signaling molecules, such as Lck, and that down-regulation of PTP-PEST may be a reason for the increased response to TCR triggering of previously activated T cells.

Response to Dasatinib after Imatinib Failure According to Type of Preexisting BCR-ABL Mutations.

Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC kinases with preclinical activity against 20/21 imatinib resistant BCR-ABL mutations and clinical phase I/II efficacy in patients with chronic myelogenous leukemia (CML) and BCR-ABL positive acute lymphoblastic leukemia (ALL). We sought to establish a relationship between type of preexisting BCR-ABL mutations associated with imatinib resistance and efficacy of dasatinib in patients (pts) with CML and ALL. We have investigated 872 peripheral blood samples from 394 pts (53% male, median age 60 yrs, range 17–85) who had been enrolled in international phase II studies investigating the activity of 70mg dasatinib BID after imatinib failure (chronic phase, CP, n=198; accelerated phase, AP, n=78; myeloid blast crisis, MyBC, n=53; lymphoid blast crisis, LyBC, or ALL, n=65).Screening for BCR-ABL mutations was performed by D-HPLC combined with DNA sequencing. During follow up, pts were monitored in 3-monthly intervals by RQ-PCR for BCR-ABL mRNA transcripts and by mutation analysis to determine the quantitative course of the preexisting mutation or the emergence of new mutations. Hematologic and cytogenetic response data have been collected sequentially for a median of 8 months (range, 2–11) after start of therapy. Prior to dasatinib, 46 different BCR-ABL mutations involving 36 amino acids were detected in 202/394 pts (51%). 162 pts showed one, 33 pts two, 6 pts three, and 1 pt four mutations. Mutations were observed in 84 pts in CP (42%), 47 pts in AP (60%), 23 pts in MyBC (43%), and 48 pts in LyBC and ALL (74%). In patients with mutations, hematologic response was 91% in CP, 62% in AP, 41% in MyBC, and 34% in LyBC/ALL (p<0.01), major and complete cytogenetic response did not differ significantly (47% and 34% in CP, 35% and 27% in AP, 33% and 28% in MyBC, 53% and 51% in LyBC/ALL). Major cytogenetic response rates were comparable in pts bearing no mutations (44%), mutations within the P-Loop (43%), SH2 domain (47%), activation loop (56%), or other sites (49%), but not for T315I (0%, p<0.001). Sorting individual pts by the underlying mutation and cellular IC50 values of dasatinib revealed clearly higher hematologic and cytogenetic response rates in those with lower IC50 values. In line with the virtual insensitivity to dasatinib in vitro, none of the 17 pts carrying a T315I mutation showed any hematologic response. Two distinct patterns of response were observed:a parallel decrease of the BCR-ABL load and the mutated clone ora decrease of the BCR-ABL load followed by a decrease of the mutated clone after a delay of up to 4–6 months.Up until now 5 pts developed new mutations associated with dasatinib resistance (T315I, n=2, AP and MyBC pts; F317L+F486S, n=2, LyBC and MyBC pts; E507G, n=1, CP pt). We conclude that dasatinib is capable of inducing hematologic and cytogenetic remissions in a significant proportion of imatinib resistant pts harboring BCR-ABL mutations, except T315I. Response dynamics depend on the individual type of mutation which may be a basis for individual dose adaptation according to the mutation pattern.

Protein kinase D2 mediates lysophosphatidic acid-induced interleukin 8 production in nontransformed human colonic epithelial cells through NF-κB

The signaling pathways mediating lysophosphatidic acid (LPA)-stimulated PKD(2) activation and the potential contribution of PKD(2) in regulating LPA-induced interleukin 8 (IL-8) secretion in nontransformed, human colonic epithelial NCM460 cells were examined. Treatment of serum-deprived NCM460 cells with LPA led to a rapid and striking activation of PKD(2), as measured by in vitro kinase assay and phosphorylation at the activation loop (Ser706/710) and autophosphorylation site (Ser876). PKD(2) activation induced by LPA was abrogated by preincubation with selective PKC inhibitors GF-I and Ro-31-8220 in a dose-dependent manner. These inhibitors did not have any direct inhibitory effect on PKD(2) activity. LPA induced a striking increase in IL-8 production and stimulated NF-kappaB activation, as measured by NF-kappaB-DNA binding, NF-kappaB-driven luciferase reporter activity, and IkappaBalpha phosphorylation. PKD(2) gene silencing utilizing small interfering RNAs targeting distinct PKD(2) sequences dramatically reduced LPA-stimulated NF-kappaB promoter activity and IL-8 production. PKD(2) activation is a novel early event in the biological action of LPA and mediates LPA-stimulated IL-8 secretion in NCM460 cells through a NF-kappaB-dependent pathway. Our results demonstrate, for the first time, the involvement of a member of the PKD family in the production of IL-8, a potent proinflammatory chemokine, by epithelial cells.

Protein Kinase D Intracellular Localization and Activity Control Kinase D-interacting Substrate of 220-kDa Traffic through a Postsynaptic Density-95/Discs Large/Zonula Occludens-1-binding Motif

Protein kinase D (PKD) controls protein traffic from the trans-Golgi network (TGN) to the plasma membrane of epithelial cells in an isoform-specific manner. However, whether the different PKD isoforms could be selectively regulating the traffic of their specific substrates remains unexplored. We identified the C terminus of the different PKDs that constitutes a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3, to be responsible for the differential control of kinase D-interacting substrate of 220-kDa (Kidins220) surface localization, a neural membrane protein identified as the first substrate of PKD1. A kinase-inactive mutant of PKD3 is only able to alter the localization of Kidins220 at the plasma membrane when its C terminus has been substituted by the PDZ-binding motif of PKD1 or PKD2. This isoform-specific regulation of Kidins220 transport might not be due to differences among kinase activity or substrate selectivity of the PKD isoenzymes but more to the adaptors bound to their unique C terminus. Furthermore, by mutating the autophosphorylation site Ser(916), located at the critical position -2 of the PDZ-binding domain within PKD1, or by phorbol ester stimulation, we demonstrate that the phosphorylation of this residue is crucial for Kidins220-regulated transport. We also discovered that Ser(916) trans-phosphorylation takes place among PKD1 molecules. Finally, we demonstrate that PKD1 association to intracellular membranes is critical to control Kidins220 traffic. Our findings reveal the molecular mechanism by which PKD localization and activity control the traffic of Kidins220, most likely by modulating the recruitment of PDZ proteins in an isoform-specific and phosphorylation-dependent manner.

Translational Deregulation in PDK-1−/− Embryonic Stem Cells

PDK-1 is a protein kinase that is critical for the activation of many downstream protein kinases in the AGC superfamily, through phosphorylation of the activation loop site on these substrates. Cells lacking PDK-1 show decreased activity of these protein kinases, including protein kinase B (PKB) and p70S6K, whereas mTOR activity remains largely unaffected. Here we show, by assessing both association of cellular RNAs with polysomes and by metabolic labeling, that PDK-1-/- embryonic stem (ES) cells exhibit defects in mRNA translation. We identify which mRNAs are most dramatically translationally regulated in cells lacking PDK-1 expression by performing microarray analysis of total and polysomal RNA in these cells. In addition to the decreased translation of many RNAs, a smaller number of RNAs show increased association with polyribosomes in PDK-1-/- ES cells relative to PDK-1+/+ ES cells. We show that PKB activity is a critical downstream component of PDK-1 in mediating translation of cystatin C, RANKL, and Rab11a, whereas mTOR activity is less important for effective translation of these targets.