KSHV-TK: thymidine kinase or tyrosine kinase?

The thymidine kinases (TK) of alphaherpesviruses phosphorylate nucleosides, allowing viral replication in non‐dividing cells. They also phosphorylate acyclovir (ACV), a specific antiviral when modified. Despite encoding a TK homolog, Kaposi's sarcoma‐associated herpesvirus (KSHV), a gammaherpesvirus, is relatively immune to the effects of ACV. In this issue, Gill et al ( 2015 ) show that rather than functioning as a thymidine kinase, the KSHV‐TK homolog has evolved a unique function as a tyrosine kinase that is autophosphorylated. KSHV‐TK autophosphorylation of three SH2 domains leads to Crk binding and likely sequestration of Crk from focal adhesions. KSHV‐TK also binds to FAK with a concurrent loss of phosphorylation in the focal adhesions, leading to a loss of cell morphology and membrane blebbing. Rather than acting to create nucleotide pools for replication, the KSHV‐TK homolog may play a pivotal role in viral pathogenesis by altering focal adhesions and cell detachment.     

Integrin adhesion: when is a kinase a kinase?

Recent genetic studies in the worm Caenorhabditis elegans and fruitfly Drosophila have revealed the essential role integrin-linked kinase plays in integrin adhesion - but it apparently acts in this role as an adaptor rather than a kinase.     

Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage-induced apoptosis

Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage-induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner.     

Diacylglycerol kinase θ counteracts protein kinase C-mediated inactivation of the EGF receptor

Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC) signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGKθ isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGKθ physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGKθ attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGKθ downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGKθ translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGKθ attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling.     

Leucine-rich repeat kinase 2 induces α-synuclein expression via the extracellular signal-regulated kinase pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes α-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.     

A role for protein kinase casein kinase2 α-subunits in the Arabidopsis circadian clock

Circadian rhythms are autoregulatory, endogenous rhythms with a period of approximately 24 h. A wide variety of physiological and molecular processes are regulated by the circadian clock in organisms ranging from bacteria to humans. Phosphorylation of clock proteins plays a critical role in generating proper circadian rhythms. Casein Kinase2 (CK2) is an evolutionarily conserved serine/threonine protein kinase composed of two catalytic α-subunits and two regulatory β-subunits. Although most of the molecular components responsible for circadian function are not conserved between kingdoms, CK2 is a well-conserved clock component modulating the stability and subcellular localization of essential clock proteins. Here, we examined the effects of a cka1a2a3 triple mutant on the Arabidopsis (Arabidopsis thaliana) circadian clock. Loss-of-function mutations in three nuclear-localized CK2α subunits result in period lengthening of various circadian output rhythms and central clock gene expression, demonstrating that the cka1a2a3 triple mutant affects the pace of the circadian clock. Additionally, the cka1a2a3 triple mutant has reduced levels of CK2 kinase activity and CIRCADIAN CLOCK ASSOCIATED1 phosphorylation in vitro. Finally, we found that the photoperiodic flowering response, which is regulated by circadian rhythms, was reduced in the cka1a2a3 triple mutant and that the plants flowered later under long-day conditions. These data demonstrate that CK2α subunits are important components of the Arabidopsis circadian system and their effects on rhythms are in part due to their phosphorylation of CIRCADIAN CLOCK ASSOCIATED1.     

Regulation of type IIα phosphatidylinositol phosphate kinase localisation by the protein kinase CK2

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIα PIP kinase at a single site unique to that isoform – Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

c-Abl tyrosine kinase regulates serum-induced nuclear export of diacylglycerol kinase α by phosphorylation at Tyr-218

c-Abl is a tyrosine kinase involved in many cellular processes, including cell cycle control and proliferation. However, little is known about its substrates. Here, we show that c-Abl directly phosphorylates diacylglycerol kinase α (DGKα), an important regulator of many cellular events through its conversion of diacylglycerol to phosphatidic acid. We found that DGKα was transported from the cytoplasm to the nucleus in response to serum starvation, and serum restoration induced the nuclear export of the enzyme to the cytoplasm. This serum-induced export involves two tyrosine kinases, c-Src and c-Abl. The latter, c-Abl, is activated by c-Src, phosphorylates DGKα, and shuttles between the nucleus and the cytoplasm in a direction opposite to that of DGKα in response to serum restoration. Moreover, an in vitro phosphorylation assay using purified mutants of DGKα identified Tyr-218 as a site of phosphorylation by c-Abl. We confirmed these results for endogenous DGKα using an antibody specific for phospho-Tyr-218, and this phosphorylation was necessary for the serum-induced export of DGKα. These results demonstrate that the nucleo-cytoplasmic shuttling of DGKα is orchestrated by tyrosine phosphorylation by the Src-activated tyrosine kinase c-Abl and that this phosphorylation is important for regulating the function of cytoplasmic and/or nuclear DGKα.     

Protein kinase Cα and Src kinase support human prostate-distributed dihydrotestosterone-metabolizing UDP-glucuronosyltransferase 2B15 activity

Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17β-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70–100 and 80–90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130–140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D₃ enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-³³P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55–58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions.     

Generation of autonomous activity of Ca(2+)/calmodulin-dependent protein kinase kinase β by autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) phosphorylate and activate specific downstream protein kinases, including CaMKI, CaMKIV, and 5'-AMP-activated protein kinase, which mediates a variety of Ca(2+) signaling cascades. CaMKKs have been shown to undergo autophosphorylation, although their role in enzymatic regulation remains unclear. Here, we found that CaMKKα and β isoforms expressed in nonstimulated transfected COS-7 cells, as well as recombinant CaMKKs expressed in and purified from Escherichia coli, were phosphorylated at Thr residues. Introduction of a kinase-dead mutation completely impaired the Thr phosphorylation of these recombinant CaMKK isoforms. In addition, wild-type recombinant CaMKKs were unable to transphosphorylate the kinase-dead mutants, suggesting that CaMKK isoforms undergo Ca(2+)/CaM-independent autophosphorylation in an intramolecular manner. Liquid chromatography-tandem mass spectrometry analysis identified Thr(482) in the autoinhibitory domain as one of the autophosphorylation sites in CaMKKβ, but phosphorylation of the equivalent Thr residue (Thr(446)) in the α isoform was not observed. Unlike CaMKKα that has high Ca(2+)/CaM-dependent activity, wild-type CaMKKβ displays enhanced autonomous activity (Ca(2+)/CaM-independent activity, 71% of total activity). This activity was significantly reduced (to 37%) by substitution of Thr(482) with a nonphosphorylatable Ala, without significant changes in Ca(2+)/CaM binding. In addition, a CaMKKα mutant containing the CaMKKβ regulatory domain was shown to be partially phosphorylated at Thr(446), resulting in a modest elevation of its autonomous activity. The combined results indicate that, in contrast to the α isoform, CaMKKβ exhibited increased autonomous activity, which was caused, at least in part, by autophosphorylation at Thr(482), resulting in partial disruption of the autoinhibitory mechanism.     

Liver phosphorylase kinase: characterization of two interconvertible forms and partial purification of phosphorylase kinase α

Summary The presence of two interconvertible forms of phosphorylase kinase has been confirmed in rat liver extracts. The pH optimum of the nonactivated form (PhK b) was lower than the pH optimum of the activated form (PhK ) as reported by others (2). In the absence of calcium the K_m of PhK for phosphorylase b was 53 + 10 U/ ml with a V_m of 17 = 1 U/gm of tissue. The K_m of PhK for phosphorylase b was 20 + 2 U/ml with a V_m of 65 U/gm. Calcium stimulated both forms of phosphorylase kinase(A_0.5 0.03 M). In the presence of 0.1 M calcium the Km for phosphorylase b of both forms of the enzyme was reduced. In addition, calcium increased the V_m of both forms, but the effect was greater for PhK b than for PhK . The K_m of both forms of phosphorylase kinase for ATP was 0.05 mM and was unaffected by calcium. All of these studies were done using liver phosphorylase b as substrate. Conditions for assaying PhK activity virtually independent of PhK b activity also are indicated. This will enable the monitoring of interconversion reactions in tissue extracts.Phosphorylase kinase a was purified to near homogeneity using DEAE-cellulose, Sepharose 4B gel filtration and ATP affinity chromatography. The molecular weight was approximately 1 × 106. The pII profile, calcium requirements and kinetic constants were the same as those for PhK a in the crude extract.     

Diacylglycerol kinase η colocalizes and interacts with apoptosis signal-regulating kinase 3 in response to osmotic shock

Diacylglycerol kinase (DGK) η translocates from the cytoplasm to punctate vehicles via osmotic shock. Apoptosis signal-regulating kinase (ASK) 3 (MAP kinase kinase kinase (MAPKKK) 15) is also reported to respond to osmotic shock. Therefore, in the present study, we examined the subcellular localization of DGKη and ASK3 expressed in COS-7 cells under osmotic stress. We found that DGKη was almost completely colocalized with ASK3 in punctate structures in response to osmotic shock. In contrast, DGKδ, which is closely related to DGKη structurally, was not colocalized with ASK3, and DGKη failed to colocalize with another MAPKKK, C-Raf, even under osmotic stress. The structures in which DGKη and ASK3 localized were not stained with stress granule makers. Notably, DGKη strongly interacted with ASK3 in an osmotic shock-dependent manner. These results indicate that DGKη and ASK3 undergo osmotic shock-dependent colocalization and associate with each other in specialized structures.     

Fluorophore labeled kinase detects ligands that bind within the MAPK insert of p38α kinase

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway.     

Identification of mitogen-activated protein kinase kinase gene family and MKK–MAPK interaction network in maize

Plant mitogen-activated protein kinases (MAPK) are involved in important processes, including stress signaling and development. MAPK kinases (MAPKK, MKK) have been investigated in several plant species including Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, and Brachypodium distachyon. In the present study, nine putative maize MKK genes have been identified. Analysis of the conserved protein motifs, exon–intron junctions and intron phase has revealed high levels of conservation within the phylogenetic groups. Next, we defined four new ZmMKK–ZmMPK interactions using yeast two-hybrid. Finally, we examined the biological functions of the ZmMKK4 gene. Overexpression of ZmMKK4 in Arabidopsis conferred tolerance to oxidative stress by increased germination rate and early seedling growth compared with WT plants. Taken together, we provide a comprehensive bioinformatics analysis of the MKK gene family in maize genome and our data provide an important foundation for further functional study of MAPK and MKK families in maize.     

Protein kinase Cζ and glucose uptake

Protein kinase Cζ (PKCζ) is a member of the PKC family, serving downstream of insulin receptor and phosphatidylinositol (PI) 3-kinase. Many evidences suggest that PKCζ plays a very important role in activating glucose transport response. Not only insulin but also glucose and exercise can activate PKCζ through diverse pathways. PKCζ activation and activity are impaired with insulin resistance in muscle and adipose tissues of type II diabetes individuals, but heightened in liver tissue, wherein it also increases lipid synthesis mediated by SREBP-1c (sterol-regulatory element-binding protein). Many studies have focused on linkage between PKCζ and GLUT4 translocation and activation. Exploring the molecular mechanisms and pathways by which PKCζ mediates glucose transport will highlight the insulin-signaling pathway. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 7, pp. 869–875. Co-first authors.     

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.     

Is creatine kinase responsible for fatigue? Studies of isolated skeletal muscle deficient in creatine kinase.

Creatine kinase (CK) is a key enzyme for maintaining a constant ATP/ADP ratio during rapid energy turnover. To investigate the role of CK in skeletal muscle fatigue, we used isolated whole muscles and intact single fibers from CK-deficient mice (CK(-/-)). With high-intensity electrical stimulation, single fibers from CK(-/-) mice displayed a transient decrease in both tetanic free myoplasmic [Ca(2+)] ([Ca(2+)](i), measured with the fluorescent dye indo-1) and force that was not observed in wild-type fibers. With less intense, repeated tetanic stimulation single fibers and EDL muscles, both of which are fast-twitch, fatigued more slowly in CK(-/-) than in wild-type mice; on the other hand, the slow-twitch soleus muscle fatigued more rapidly in CK(-/-) mice. In single wild-type fibers, tetanic force decreased and [Ca(2+)](i) increased during the first 10 fatiguing tetani, but this was not observed in CK(-/-) fibers. Fatigue was not accompanied by phosphocreatine breakdown and accumulation of inorganic phosphate in CK(-/-) muscles. In conclusion, CK is important for avoiding fatigue at the onset of high-intensity stimulation. However, during more prolonged stimulation, CK may contribute to the fatigue process by increasing the myoplasmic concentration of inorganic phosphate.