Crystal structure and allosteric activation of protein kinase C βII

Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C βII was determined at 4.0 ?, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCβII was derived from small-angle X-ray scattering. Together, these results show how PKCβII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism.     

Diacylglycerol activation of protein kinase Cε and hepatic insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is now the most frequent chronic liver disease in Western societies, affecting one in four adults in the USA, and is strongly associated with hepatic insulin resistance, a major risk factor in the pathogenesis of type 2 diabetes. Although the cellular mechanisms underlying this relationship are unknown, hepatic accumulation of diacylglycerol (DAG) in both animals and humans has been linked to hepatic insulin resistance. In this Perspective, we discuss the role of DAG activation of protein kinase Cε as the mechanism responsible for NAFLD-associated hepatic insulin resistance seen in obesity, type 2 diabetes, and lipodystrophy.     

Role of proteolytic activation of protein kinase Cδ in the pathogenesis of prion disease

Prion diseases are infectious and inevitably fatal neurodegenerative diseases characterized by prion replication, widespread protein aggregation and spongiform degeneration of major brain regions controlling motor function. Oxidative stress has been implicated in prion-related neuronal degeneration, but the molecular mechanisms underlying prion-induced oxidative damage are not well understood. In this study, we evaluated the role of oxidative stress-sensitive, pro-apoptotic protein kinase Cδ (PKCδ) in prion-induced neuronal cell death using cerebellar organotypic slice cultures (COSC) and mouse models of prion diseases. We found a significant upregulation of PKCδ in RML scrapie-infected COSC, as evidenced by increased levels of both PKCδ protein and its mRNA. We also found an enhanced regulatory phosphorylation of PKCδ at its two regulatory sites, Thr505 in the activation loop and Tyr311 at the caspase-3 cleavage site. The prion infection also induced proteolytic activation of PKCδ in our COSC model. Immunohistochemical analysis of scrapie-infected COSC revealed loss of PKCδ positive Purkinje cells and enhanced astrocyte proliferation. Further examination of PKCδ signaling in the RML scrapie adopted in vivo mouse model showed increased proteolytic cleavage and Tyr 311 phosphorylation of the kinase. Notably, we observed a delayed onset of scrapie-induced motor symptoms in PKCδ knockout (PKCδ^−/−) mice as compared with wild-type (PKCδ^+/+) mice, further substantiating the role of PKCδ in prion disease. Collectively, these data suggest that PKCδ signaling likely plays a role in the neurodegenerative processes associated with prion diseases.     

Caspase-mediated activation of PAK2 during apoptosis: proteolytic kinase activation as a general mechanism of apoptotic signal transduction?

p21-activated kinase 2 (PAK2) is proteolytically cleaved during apoptosis through the action of DEVD-sensitive caspase(s). This cleavage event causes PAK2 activation, and PAK2 activity is implicated in regulation of the biochemistry and morphology of the apoptotic cell. PAK2 is just one example of a number of identified caspase targets that are protein kinases involved in regulating various aspects of cell function. We hypothesize that this may reflect their important role in regulating the controlled and orderly demise of the dying cell.     

Glucose and fatty acids synergize to promote B-cell apoptosis through activation of glycogen synthase kinase 3β independent of JNK activation

Background

The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic β-cell death. This study examines the synergistic effects of glucose and FFA on β-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined.

Principal Findings

Increasing glucose (5–25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3β (Gsk3β) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3β.

Conclusions/Significance

Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3β activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet β-cell Gsk3β as a potential therapeutic target.     

Is receptor oligomerization causally linked to activation of the EGF receptor kinase?

Transduction of a signal from an extracellular peptide hormone to produce an intracellular response is often mediated by a cell surface receptor, which is usually a glycoprotein. The secondary intracellular signal(s) generated after hormone binding to the receptor have been intensively studied. The nature of the primary signal generated by ligand binding to the receptor is understood less well in most cases. The particular case of the epidermal growth factor (EGF) receptor is analyzed, and evidence for or against two dissimilar models of primary signal transduction is reviewed. Evidence for the most widely accepted current model is found to be unconvincing. Evidence for the other model is substantial but indirect; a direct test of this model remains to be done.     

Beyond NF-κB activation: nuclear functions of IκB kinase α

IκB kinase (IKK) complex, the master kinase for NF-κB activation, contains two kinase subunits, IKKα and IKKβ. In addition to mediating NF-κB signaling by phosphorylating IκB proteins during inflammatory and immune responses, the activation of the IKK complex also responds to various stimuli to regulate diverse functions independently of NF-κB. Although these two kinases share structural and biochemical similarities, different sub-cellular localization and phosphorylation targets between IKKα and IKKβ account for their distinct physiological and pathological roles. While IKKβ is predominantly cytoplasmic, IKKα has been found to shuttle between the cytoplasm and the nucleus. The nuclear-specific roles of IKKα have brought increasing complexity to its biological function. This review highlights major advances in the studies of the nuclear functions of IKKα and the mechanisms of IKKα nuclear translocation. Understanding the nuclear activity is essential for targeting IKKα for therapeutics.     

Subcellular distribution and activation of rat submandibular cAMP-dependent protein kinase following β-adrenergic receptor stimulation

The extent of activation of rat submandibular protein kinase A (EC 2.7.1.37) isozymes following β-adrenergic receptor stimulation was determined in vitro using dispersed cells and an 8-N₃-[³²P]cAMP photoprobe. The half-maximal binding of the photoprobe for microsomal and cytosolic type I and cytosolic type II was 9 nM, 27 nM and 92 nM, respectively. ‘Cold trap’ studies indicated that 70% of type I protein kinase A was activated following maximal β-adrenergic receptor stimulation, whereas type II activation was less than 40%. Both cytosolic and microsomal type I activation occurred rapidly following β-adrenergic receptor stimulation and both remain activated throughout the entire secretory period. Type I inactivation occurred rapidly subsequent to β-adrenergic receptor blockade. The dose-response relationship for the isotypes following β-adrenergic receptor activation demonstrated a greater extent of type I activation at submaximal concentrations of agonist. Although protein kinase A may not be the only kinase involved in rat submandibular mucin release, these data add further support to a direct regulatory role for this kinase, with type I having potentially a greater role than type II.     

α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A)-adrenergic receptor (α(1A)-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α(1A)-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α(1A)-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α(1A)-AR. α(1A)-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α(1A)-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31-8220 (a PKC inhibitor) inhibited α(1B)-AR- but not α(1A)-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α(1A)-AR-induced ERK1/2 activation, which is independent of G(q)/PLC/PKC signaling.     

Role of ��-tocopherol in cellular signaling: ��-tocopherol inhibits stress-induced mitogen-activated protein kinase activation

Tocopherols belong to the plant-derived poly phenolic compounds known for antioxidant functions in plants and animals. Activation of mitogen-activated protein kinases (MAPK) is a common reaction of plant cells in defense-related signal transduction pathways. We report a novel non-antioxidant function of α-tocopherol in higher plants linking the physiological role of tocopherol with stress signalling pathways. Pre-incubation of a low concentration of 50 μM α-tocopherol negatively interferes with MAPK activation in elicitor-treated tobacco BY2 suspension culture cells and wounded tobacco leaves, whereas pre-incubated BY2 cells with α-tocopherol phosphate did not show the inhibitory effect on stimuli-induced MAPK activation. The decreased MAPK activity was neither due to a direct inhibitory effect of α-tocopherol nor due to the induction of an inhibitory or inactivating activity directly affecting MAPK activity. The data support that the target of α-tocopherol negatively regulates an upstream component of the signaling pathways that leads to stress dependent MAPK activation.     

Nitric oxide-induced activation of the AMP-activated protein kinase α2 subunit attenuates IκB kinase activity and inflammatory responses in endothelial cells

Background

In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFκB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO).

Methodology/Principal Findings

Overexpression of a dominant negative AMPKα2 mutant in tumor necrosis factor-α-stimulated human endothelial cells resulted in increased NFκB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKα2^-/- mice the interleukin (IL)-1β induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFκB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKα2^-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IκB and p65, indicating a link between AMPK and the IκB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKα2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKα2^+/+ versus AMPKα2^-/- mice.

Conclusions

These results demonstrate that the IKK is a direct substrate of AMPKα2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFκB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.     

Synapses of amphids defective (SAD-A) kinase promotes glucose-stimulated insulin secretion through activation of p21-activated kinase (PAK1) in pancreatic β-Cells

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis.     

Ala99ser mutation in RIα Regulatory Subunit of protein kinase A causes reduced kinase activation by cAMP and arrest of hormone-dependent breast cancer cell growth

Expression of the RI regulatory subunit of protein kinase A type I is increased in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. Ala99 (the pseudophosphorylation site) of human RI was replaced with Ser (RI-p) for the structure-function analysis of RI. MCF-7 hormone- dependent breast cancer cells were transfected with an expression vector for the wild-type RI or mutant RI-p. Overexpression of RI-P resulted in suppression of protein kinase A type II, the isozyme of type I kinase, production of kinase exhibiting reduced cAMP activation, and inhibition of cell growth showing an increase in G0/G1 phase of the cell cycle and apoptosis. The wild-type RI overexpression had no effect on protein kinase A isozyme distribution or cell growth. Overexpression of protein kinase A type II regulatory subunit, RII, suppressed RI and protein kinase A type I and inhibited cell growth. These results show that the growth of hormone-dependent breast cancer cells is dependent on the functional protein kinase A type I.     

Phospholipase C and protein kinase C-β 2 mediate insulin-like growth factor II-dependent sphingosine kinase 1 activation

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.