Tropomyosin Tm5NM1 spatially restricts src kinase activity through perturbation of rab11 vesicle trafficking

In order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules.

Inhibiting Eph kinase activity may not be “Eph”ective for cancer treatment

Several Eph receptor tyrosine kinases (RTKs) are commonly over-expressed in epithelial and mesenchymal cancers and are recognized as promising therapeutic targets. Although normal interaction between Eph receptors and their ephrin ligands stimulates kinase activity and is generally tumor suppressive, significant Eph over-expression allows activation of ligand- and/or kinase-independent signaling pathways that promote oncogenesis. Single-agent kinase inhibitors are widely used to target RTK-driven tumors but acquired and de novo resistance to such agents is a major limitation to effective clinical use. Accumulating evidence suggests that Ephs can be inhibited by “leaky” or low-specificity kinase inhibitors targeted at other RTKs. Such off-target effects may therefore inadvertently promote ligand- and/or kinase-independent oncogenic Eph signaling, thereby providing a new mechanism by which resistance to the RTK inhibitors can emerge. We propose that combining specific, non-leaky kinase inhibitors with tumor-suppressive stimulators of Eph signaling may provide more effective treatment options for overcoming treatment-induced resistance and clinical failure.

Targeting aberrant kinase activity in myeloid leukaemias

Acute myeloid leukaemia (AML) is the most common form of acute leukaemia in adults. Its treatment has remained largely unchanged for the past 30 years. Chronic myeloid leukaemia (CML) represents a tremendous success story in the era of targeted therapy but significant challenges remain including the development of drug resistance and disease persistence due to presence of CML stem cells. The Aurora family of kinases is essential for cell cycle regulation and their aberrant expression in cancer prompted the development of small molecules that selectively inhibit their activity. Chapter 2 of this thesis outlines the efficacy and mechanism of action of alisertib, a novel inhibitor of Aurora A kinase, in preclinical models of CML. Alisertib possessed equipotent activity against CML cells expressing unmutated and mutated forms of BCR-ABL. Notably, this agent retained high activity against the T315I and E255K BCR-ABL mutations, which confer the greatest degree of resistance to standard CML therapy. Chapter 3 explores the activity of alisertib in preclinical models of AML. Alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the forkhead box O3 (FOXO3a) targets p27 and BCL-2 interacting mediator (BIM), and triggered apoptosis. A link between Aurora A expression and sensitivity to ara-C was established. Chapter 4 outlines the role of the proto-oncogene serine/threonine-protein (PIM) kinases in resistance to ara-C in AML. We report that the novel small molecule PIM kinase inhibitor SGI-1776 disrupted cell viability and induced apoptosis in AML. We establish a link between ara-C resistance and PIM over-expression. Finally, chapter 5 explores how the preclinical work outlined in this thesis may be translated into clinical studies that may lead to novel therapeutic approaches for patients with refractory myeloid leukaemia.

In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.

BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.

Regulation of DLK-1 kinase activity by calcium-mediated dissociation from an inhibitory isoform.

MAPKKK dual leucine zipper-bearing kinases (DLKs) are regulators of synaptic development and axon regeneration. The mechanisms underlying their activation are not fully understood. Here, we show that C. elegans DLK-1 is activated by a Ca(2+)-dependent switch from inactive heteromeric to active homomeric protein complexes. We identify a DLK-1 isoform, DLK-1S, that shares identical kinase and leucine zipper domains with the previously described long isoform DLK-1L but acts to inhibit DLK-1 function by binding to DLK-1L. The switch between homo- or heteromeric DLK-1 complexes is influenced by Ca(2+) concentration. A conserved hexapeptide in the DLK-1L C terminus is essential for DLK-1 activity and is required for Ca(2+) regulation. The mammalian DLK-1 homolog MAP3K13 contains an identical C-terminal hexapeptide and can functionally complement dlk-1 mutants, suggesting that the DLK activation mechanism is conserved. The DLK activation mechanism is ideally suited for rapid and spatially controlled signal transduction in response to axonal injury and synaptic activity.

CDK11(p58) protein kinase activity is associated with Bcl-2 down-regulation in pro-apoptosis pathway.

CDK11(p58), a G2/M-specific protein kinase, has been shown to be associated with apoptosis in many cell lines, with largely unknown mechanisms. Our previous study proved that CDK11(p58)-enhanced cycloheximide (CHX)-induced apoptosis in SMMC-7721 hepatocarcinoma cells. Here we report for the first time that ectopic expression of CDK11(p58) down-regulates Bcl-2 expression and its Ser70, Ser87 phosphorylation in CHX-induced apoptosis in SMMC-7721 cells. Overexpression of Bcl-2 counteracts the pro-apoptotic activity of CDK11(p58). Furthermore, we confirm that the kinase activity of CDK11(p58) is essential to the down-regulation of Bcl-2 as well as apoptosis. Taken together, these results demonstrate that CDK11(p58) down-regulates Bcl-2 in pro-apoptosis pathway depending on its kinase activity, which elicits survival signal in hepatocarcinoma cells.

A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia

Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis. We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction. The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate. The development of a protocol for the physical recovery of polyP from solution is also reported. (C) 2002 Elsevier Science (USA). All rights reserved.

Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased similar to6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.

The respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen-activated protein kinase p38-dependent tyrosine kinase activity during virus infection

The phosphorylation status of the small hydrophobic (SH) protein of respiratory syncytial virus (RSV) was examined in virus-infected Vero cells. The SH protein v.,as isolated from [S-35]methionine- and [P-33]orthophosphate-labelled IRSV-infected cells and analysed by SDS-PAGE. In each case, a protein product of the expected size for the SH protein was observed. Phosphoamino acid analysis and reactivity with the phosphotyrosine specific antibody PY20 showed that the SH protein was modified by tyrosine phosphorylation. The role or tyrosine kinase activity in SH protein phosphorylation was confirmed by the use of genistein, a broad-spectrum tyrosine kinase inhibitor, to inhibit SH protein phosphorylation. Further analysis showed that the different glycosylated forms of the SH protein were phosphorylated, as was the oligomeric form of the protein. Phosphorylation of the SH protein was specifically inhibited by the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580, suggesting that SH protein phosphorylation occurs via a MAPK p38-dependent pathway. Analysis of virus-infected cells using fluorescence microscopy showed that, although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate, at low levels, in the endoplasmic reticulum/Golgi complex, confirming recent observations. However, in the presence of SB203580. an increased accumulation of the SH protein in the Golgi complex was observed, although other virus structures, such as virus filaments and inclusion bodies, remained largely unaffected. These results showed that during RSV infection, the SH protein is modified by an MAPK p38-dependant tyrosine kinase activity and that this modification influences its cellular distribution.

Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

Inhibition of LRRK2 kinase activity stimulates macroautophagy

Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific target for disease modifying therapies in patients.

GRID and docking analyses reveal a molecular basis for flavonoid inhibition of src-family kinase activity

Flavonoids reduce cardiovascular disease risk through anti-inflammatory, anti-coagulant and anti-platelet actions. One key flavonoid inhibitory mechanism is blocking kinase activity that drives these processes. Flavonoids attenuate activities of kinases including phosphoinositide-3-kinase (PI3K), Fyn, Lyn, Src, Syk, PKC, PIM1/2, ERK, JNK, and PKA. X-ray crystallographic analyses of kinase-flavonoid complexes show that flavonoid ring systems and their hydroxyl substitutions are important structural features for their binding to kinases. A clearer understanding of structural interactions of flavonoids with kinases is necessary to allow construction of more potent and selective counterparts. We examined flavonoid (quercetin, apigenin and catechin) interactions with Src-family kinases (Lyn, Fyn and Hck) applying the Sybyl docking algorithm and GRID. A homology model (Lyn) was used in our analyses to demonstrate that high quality predicted kinase structures are suitable for flavonoid computational studies. Our docking results revealed potential hydrogen bond contacts between flavonoid hydroxyls and kinase catalytic site residues. Identification of plausible contacts indicated that quercetin formed the most energetically stable interactions, apigenin lacked hydroxyl groups necessary for important contacts, and the non-planar structure of catechin could not support predicted hydrogen bonding patterns. GRID analysis using a hydroxyl functional group supported docking results. Based on these findings, we predicted that quercetin would inhibit activities of Src-family kinases with greater potency than apigenin and catechin. We validated this prediction using in vitro kinase assays. We conclude that our study can be used as a basis to construct virtual flavonoid interaction libraries to guide drug discovery using these compounds as molecular templates.