Regulation of Plasmodium falciparum Pfnek3 relies on phosphorylation at its activation loop and at threonine 82

A mitogen-activated protein kinase (MAPK), Pfmap2, has been identified in Plasmodium falciparum. However, its bona fide activator remains elusive as no MAPK kinase (MAPKK) homologues have been found so far. Instead, Pfnek3, a NIMA (never in mitosis, Aspergillus)-related kinase, was earlier reported to display a MAPKK-like activity due to its activating effect on Pfmap2. In this study, the regulatory mechanism of Pfnek3 was investigated. Pfnek3 was found to possess a SSEQSS motif within its activation loop that fulfills the consensus SXXXS/T phospho-activating sequence of MAPKKs. Functional analyses of the SSEQSS motif by site-directed mutagenesis revealed that phosphorylation of residues S221 and S226 is essential for mediating Pfnek3 activity. Moreover, via tandem mass-spectrometry, residue T82 was uncovered as an additional phosphorylation site involved in Pfnek3 activation. Collectively, these results provide valuable insights into the potential in vivo regulation of Pfnek3, with residues T82, S221 and S226 functioning as phospho-activating sites.     

Inhibition of invasion and intraerythrocytic development ofPlasmodium falciparum by kinase inhibitors

We have examined the effects of seven protein kinase inhibitors (staurosporine, genistein, methyl 2,5-dihydroxycinnamate, tyrphostins B44 and B46, lavendustin A and R03) on the erythrocytic cycle of the malaria parasite,Plasmodium falciparum. One (staurosporine) strongly inhibits serine/threonine kinases, but the remainder all exhibit a strong preference for tyrosine kinases. We have been able to discriminate between effects on invasion and on intraerythrocytic development. All reagents impeded development of intraerythrocytic parasites, though at widely differing concentrations, from the sub-micromolar to the millimolar. Several inhibitors, including staurosporine, also reduced invasion. The phosphatase inhibitor, okadaic acid, had a strong inhibitory effect both on invasion and development. The regulation of malaria development by phosphorylation or dephosphorylation reactions at several points in the blood-stage cycle is implied.     

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H₂O₂, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase α chain is probably tyrosine-phosphorylated, but demonstrated that the β chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.Received 7 January 2005; received after revision 19 April 2005; accepted 22 April 2005     

A “SYDE” effect of hierarchical phosphorylation: possible relevance to the cystic fibrosis basic defect

The motif “SYDE”, incorporating the protein kinase CK2 consensus sequence (S-x-x-E) has been found to be phosphorylated at both its serine and tyrosine residues in several proteins. Of special interest is the case of cystic fibrosis Transmembrane-conductance Regulator (CFTR), where this motif is close to the residue (F508), whose deletion is the by far commonest cause of cystic fibrosis. Intriguingly, however, CFTR S511 cannot be phosphorylated by CK2 to any appreciable extent. Using a number of peptide substrates encompassing the CFTR “SYDE” site we have recently shown that: (1) failure of CK2 to phosphorylate the S⁵¹¹YDE motif is due to the presence of Y512; (2) CK2 readily phosphorylates S511 if Y512 is replaced by a phospho-tyrosine; (3) the Src family protein tyrosine kinase Lyn phosphorylates Y512 in a manner that is enhanced by the deletion of F508. These data, in conjunction with the recent observation that by inhibiting CK2 the degradation of F508delCFTR is reduced, lead us to hypothesize that the hierarchical phosphorylation of the motif SYDE by the concerted action of protein tyrosine kinases and CK2 is one of the mechanisms that cooperate to the premature degradation of F508delCFTR.     

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal “sterile α-motif” domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.     

Expression of the Stp1 LMW-PTP and inhibition of protein CK2 display a cooperative effect on immunophilin Fpr3 tyrosine phosphorylation and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> growth

Although the yeast genome does not encode bona fide protein tyrosine kinases, tyrosine-phosphorylated proteins are numerous, suggesting that besides dual-specificity kinases, some Ser/Thr kinases are also committed to tyrosine phosphorylation in Saccharomyces cerevisiae. Here we show that blockage of the highly pleiotropic Ser/Thr kinase CK2 with a specific inhibitor synergizes with the overexpression of Stp1 low-molecular-weight protein tyrosine phosphatase (PTP) in inducing a severe growth-defective phenotype, consistent with a prominent role for CK2 in tyrosine phosphorylation in yeast. We also present in vivo evidence that immunophilin Fpr3, the only tyrosine-phosphorylated CK2 substrate recognized so far, interacts with and is dephosphorylated by Spt1. These data disclose a functional correlation between CK2 and LMW-PTPs, and suggest that reversible phosphorylation of Fpr3 plays a role in the regulation of growth rate and budding in S. cerevisiae.Received 15 January 2004; received after revision 20 February 2004; accepted 4 March 2004     

Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.     

The alpha-kinase family: an exceptional branch on the protein kinase tree

The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in the context of an alpha-helix. Although recent studies show that some members of this family can also phosphorylate residues in non-helical regions, the name alpha-kinase has remained. During evolution, the alpha-kinase domains combined with many different functional subdomains such as von Willebrand factor-like motifs (vWKa) and even cation channels (TRPM6 and TRPM7). As a result, these kinases are implicated in a large variety of cellular processes such as protein translation, Mg²⁺ homeostasis, intracellular transport, cell migration, adhesion, and proliferation. Here, we review the current state of knowledge on different members of this kinase family and discuss the potential use of alpha-kinases as drug targets in diseases such as cancer.     

The expansion of GPCR transactivation-dependent signalling to include serine/threonine kinase receptors represents a new cell signalling frontier

G protein-coupled receptor (GPCR) signalling is mediated through transactivation-independent signalling pathways or the transactivation of protein tyrosine kinase receptors and the recently reported activation of the serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Since the original observation of GPCR transactivation of protein tyrosine kinase receptors, there has been considerable work on the mechanism of transactivation and several pathways are prominent. These pathways include the “triple membrane bypass” pathway and the generation of reactive oxygen species. The recent recognition of GPCR transactivation of serine/threonine kinase receptors enormously broadens the GPCR signalling paradigm. It may be predicted that the transactivation of serine/threonine kinase receptors would have mechanistic similarities with transactivation of tyrosine kinase pathways; however, initial studies suggest that these two transactivation pathways are mechanistically distinct. Important questions are the relative importance of tyrosine and serine/threonine transactivation pathways, the contribution of transactivation to overall GPCR signalling, mechanisms of transactivation and the range of cell types in which this phenomenon occurs. The ultimate significance of transactivation-dependent signalling remains to be defined but it appears to be prominent and if so will represent a new cell signalling frontier.     

Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, ³²P-poly(Glu-Tyr)_4:1 and ³²P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.Received 17 May 2004; received after revision 20 July 2004; accepted 26 July 2004     

AMP-activated protein kinase in skeletal muscle: From structure and localization to its role as a master regulator of cellular metabolism

The AMP-activated protein kinase (AMPK) is a metabolite sensing serine/threonine kinase that has been termed the master regulator of cellular energy metabolism due to its numerous roles in the regulation of glucose, lipid, and protein metabolism. In this review, we first summarize the current literature on a number of important aspects of AMPK in skeletal muscle. These include the following: (1) the structural components of the three AMPK subunits (i.e. AMPKα, β, and γ), and their differential localization in response to stimulation in muscle; (2) the biochemical regulation of AMPK by AMP, protein phosphatases, and its three known upstream kinases, LKB1, Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK), and transforming growth factor-β-activated kinase 1 (TAK1); (3) the pharmacological agents that are currently available for the activation and inhibition of AMPK; (4) the physiological stimuli that activate AMPK in muscle; and (5) the metabolic processes that AMPK regulates in skeletal muscle. Received 04 May 2008; received after revision 14 June 2008; accepted 14 July 2008     

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

Cytonuclear signaling is essential for long-term alterations of cellular properties. Several pathways involving regulated nuclear accumulation of Ser/Thr kinases have been described but little is known about cytonuclear trafficking of tyrosine kinases. Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic non-receptor tyrosine kinase enriched in neurons and involved in functions ranging from synaptic plasticity to bone resorption, as well as in cancer. We previously showed the Ca²⁺-induced, calcineurin-dependent, nuclear localization of Pyk2. Here, we characterize the molecular mechanisms of Pyk2 cytonuclear localization in transfected PC12 cells. The 700–841 linker region of Pyk2 recapitulates its depolarization-induced nuclear accumulation. This region includes a nuclear export motif regulated by phosphorylation at residue S778, a substrate of cAMP-dependent protein kinase and calcineurin. Nuclear import is controlled by a previously identified sequence in the N-terminal domain and by a novel nuclear targeting signal in the linker region. Regulation of cytonuclear trafficking is independent of Pyk2 activity. The region regulating nuclear localization is absent from the non-neuronal shorter splice isoform of Pyk2. Our results elucidate the mechanisms of Ca²⁺-induced nuclear accumulation of Pyk2. They also suggest that Pyk2 nuclear accumulation is a novel type of signaling response that may contribute to specific long-term adaptations in neurons.     

Catestatin, an endogenous Chromogranin A-derived peptide, inhibits in vitro growth of Plasmodium falciparum

Catestatin, an endogenous peptide derived from bovine chromogranin A, and its active domain cateslytin display powerful antimicrobial activities. We have tested the activities of catestatin and other related peptides on the growth of Plasmodium falciparum in vitro. Catestatin inhibits growth of the chloroquine-sensitive strain of P. falciparum 3D7, exhibiting 88% inhibition at 20 μM. A similar partial inhibition of parasite growth was observed for the chloroquine-resistant strain, 7G8 (64%,) and the multidrug-resistant strain, W2 (62%). In the presence of parasite-specific lactate dehydrogenase, a specific protein–protein interaction between catestatin and plasmepsin II precursor was demonstrated. In addition, catestatin partially inhibited the parasite-specific proteases plasmepsin in vitro. A specific interaction between catestatin and plasmepsins II and IV from P. falciparum and plasmepsin IV from the three remaining species of Plasmodium known to infect man was observed, suggesting a catestatin-induced reduction in availability of nutrients for protein synthesis in the parasite.     

Transforming growth factor-β-inducible early response gene 1 is a novel substrate for atypical protein kinase Cs

The protein kinase C (PKC) family of serine/threonine kinases consists of ten different isoforms grouped into three subfamilies, denoted classical, novel and atypical PKCs (aPKCs). The aPKCs, PKCι/λ and PKCζ serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to identify novel interaction partners for aPKCs, we performed a yeast two-hybrid screen with the regulatory domain of PKCι/λ as bait and identified the Krüppel-like factors family protein TIEG1 as a putative interaction partner for PKCι/λ. We confirmed the interaction of both aPKCs with TIEG1 in vitro and in cells, and found that both aPKCs phosphorylate the DNA-binding domain of TIEG1 on two critical residues. Interestingly, the aPKC-mediated phosphorylation of TIEG1 affected its DNA-binding activity, subnuclear localization and transactivation potential.     

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N ⁶,O ²-dibutyryl adenosine 3′5′-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12–72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35–110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus. Received 21 September 1998; received after revision 24 November 1998; accepted 3 December 1998     

Characterization of free amino acids in the hemolymph ofAchaea janata L. larvae (Lep.Noctuidae)

Summary 19 free amino acids were separated and quantified from the hemolymph of 5th instar larvae of the mothAchaea janata. Proline, histidine, threonine/glutamine/asparagine, lysine, valine and serine were the predominant amino acids in the hemolymph. Changes in amino acid concentration are discussed in relation to metabolic and other physiological activities.Acknowledgment. Y.P. Ramdev wishes to thank the Director, Indian Agricultural Research Institute, New Delhi, India for providing Senior Research Fellowship.     

Splenic B-cell activation in lipopolysaccharide-non-responsive C3H/HeJ mice by lipopolysaccharide ofPorphyromonas gingivalis

Porphyromonas gingivalis 381 lipopolysaccharide (LPS) definitely exhibited mitogenic activity in purified B-cells, separated from spleens of LPS-responsive C3H/HeN mice and LPS-non-responsive C3H/HeJ mice by using a magnetic cell sorting system. The mitogenic activity induced byP. gingivalis LPS was incompletely inhibited by polymyxin B.P. gingivalis LPS also induced a higher production of interleukin-6 (IL-6) in splenic B-cells of C3H/HeN mice as compared withEscherichia coli LPS. Furthermore,P. gingivalis LPS, but notE. coli LPS, induced definite IL-6 production in C3H/HeJ mice.P. gingivalis LPS increased tyrosine, serine/threonine phosphorylation of proteins with various major induced bands in splenic B-cells of both C3H/HeN and C3H/HeJ mice. Additionally, radioiodinatedP. gingivalis LPS, similarly toE. coli LPS, bound to a 73-kDa protein on C3H/HeJ as well as C3H/HeN B-cells. ThusP. gingivalis LPS may activate B-cells of C3H/HeJ as well as C3H/HeN mice via the LPS-specific binding protein on the cells.     

TTDN1 is a Plk1-interacting protein involved in maintenance of cell cycle integrity

Polo-like kinase 1 (Plk1) is a highly conserved serine/threonine kinase that plays critical roles in many cell cycle events, especially in mitosis. In the present study, we identified TTDN1 as a potential interacting partner of Plk1 in yeast two-hybrid screens. Sequence analysis indicates that TTDN1 contains a consensus Plk1-binding motif at its C terminus. TTDN1 colocalizes with Plk1 at the centrosome in mitosis and the midbody during cytokinesis. TTDN1 is phosphorylated by Cdk1 in mitosis, and this is required for its interaction with Plk1. Site-directed mutagenesis indicates that TTDN1 is phosphorylated at multiple residues, including Ser93 and Ser104. Mutation of Thr120 of TTDN1 abolishes its interaction with Plk1, suggesting phosphorylation of Thr120 in the consensus Plk1-binding motif is required for its interaction with Plk1. Overexpression of TTDN1 or its knockdown by siRNA causes multi-polar spindles and multiple nuclei, suggesting that TTDN1 plays a role in regulating mitosis and cytokinesis. Received 27 November 2006; received after revision 4 January 2007; accepted 25 January 2007 Y. Zhang, Y. Tian: These authors contribute equally to this work.     

Signalling via caveolin: involvement in the cross-talk between phosphoinositolglycans and insulin

In recent years, a number of cross-talk systems have been identified which feed into the insulin signalling cascade at the level of insulin receptor substrate (IRS) tyrosine phosphorylation, e.g., receptor and non-receptor tyrosine kinases and G-protein-coupled receptors. At the molecular level, a number of negative modulator and feedback systems somehow interacting with the beta-subunit (catecholamine-, phorbolester-, or tumor necrosis factor-alpha-induced serine/threonine phosphorylation, carboxy-terminal trimming by a thiol-dependent protease, association of inhibitory/regulatory proteins such as RAD, PC1, PED, alpha2-HS-glycoprotein) have been identified as candidate mechanisms for the impairment of insulin receptor function by elevations in the activity and/or amount of the corresponding modification enzymes/inhibitors. Both decreased responsiveness and sensitivity of the insulin receptor beta-subunit for insulin-induced tyrosine autophosphorylation have been demonstrated in several cellular and animal models of metabolic insulin resistance as well as in the adipose tissue and skeletal muscle of diabetic patients and obese Pima Indians compared to non-obese subjects. Therefore, induction of the insulin signalling cascade by bypassing the defective insulin receptor kinase may be useful for the therapy of non-insulin dependent diabetes mellitus. During the past two decades, phosphoinositolglycans (PIGs) of various origin have been demonstrated to exert potent insulin-mimetic metabolic effects upon incubation with cultured or isolated muscle and adipose cells. However, it remained to be elucidated whether these compounds actually manage to trigger insulin signalling and if so at which level of hierarchy within the signalling cascade the site of interference is located. Recent studies using isolated rat adipocytes and chemically synthesized PIG compounds point to IRS1/3 tyrosine phosphorylation by p59Lyn kinase as the site of cross-talk, the negative regulation of which by interaction with caveolin is apparently abrogated by PIG. This putative mechanism is thus compatible with the recently formulated caveolin signalling hypothesis, the supporting data for which are reviewed here. Though we have not obtained experimental evidence for the involvement of PIG in physiological insulin action, the potential cross-talk between insulin and PIG signalling, including the caveolae/detergent-insoluble glycolipid-enriched rafts as the compartments where the corresponding signalling components are concentrated, thus represent novel targets for signal transduction therapy.     

Isoform specific phosphorylation of p53 by protein kinase CK1

The ability of three isoforms of protein kinase CK1 (α, γ₁, and δ) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1–28 sequence. Both substrates are readily phosphoylated by CK1δ and CK1α, but not by the γ isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K _m 0.82 μM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K²²¹RQK²²⁴ loop according to modeling and mutational analysis.