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.     

TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions

The channel kinases TRPM6 and TRPM7 are both members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions and that each channel contributes uniquely to the regulation of Mg²⁺ homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other’s cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg²⁺-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg²⁺, but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wild-type TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7; however, co-expression of a TRPM6 kinase dead mutant had no effect—a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular trafficking and TRPM7-dependent cell growth. All these effects are dependent upon the presence of an active TRPM6 kinase domain. Dysregulated Mg²⁺-homeostasis causes or exacerbates many pathologies. As TRPM6 and TRPM7 are expressed simultaneously in numerous cell types, understanding how their relationship impacts regulation of Mg²⁺-uptake is thus important knowledge.     

TRPM7 is regulated by halides through its kinase domain

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg²⁺), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg²⁺ ([Mg²⁺]_i) and this is facilitated through the ATP-binding site of the channel’s kinase domain. The synergistic block of TRPM7 by chloride and Mg²⁺ is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg²⁺]_i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.     

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.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> possesses a unique dual-specificity serine/threonine and tyrosine kinase,Pfnek3

Despite the absence of classical tyrosine kinases encrypted in the kinome of Plasmodium falciparum, biochemical analyses have detected significant tyrosine phosphorylation in its cell lysates. Supporting such phosphorylation is critical for parasite development. These observations have thus raised queries regarding the plasmodial enzymes accountable for tyrosine kinase activities in vivo. In the current investigation, immunoblot analysis intriguingly demonstrated that Pfnek3, a plasmodial mitogen-activated protein kinase kinase (MAPKK), displayed both serine/threonine and tyrosine kinase activities in autophosphorylation reactions as well as in phosphorylation of the exogenous myelin basic protein substrate. The results obtained strongly support Pfnek3 as a novel dual-specificity kinase of the malarial parasite, even though it displays a HGDLKSTN motif in the catalytic loop that resembles the consensus HRDLKxxN signature found in the serine/threonine kinases. Notably, its serine/threonine and tyrosine kinase activities were found to be distinctly influenced by Mg²⁺ and Mn²⁺ cofactors. Further probing into the regulatory mechanism of Pfnek3 also revealed tyrosine phosphorylation to be a crucial factor that stimulates its kinase activity. Through biocomputational analyses and functional assays, tyrosine residues Y117, Y122, Y172, and Y238 were proposed as phosphorylation sites essential for mediating the catalytic activities of Pfnek3. The discovery of Pfnek3’s dual role in phosphorylation marks its importance in closing the loop for cellular regulation in P. falciparum, which remains elusive to date.     

Amyloid precursor protein and its homologues: a family of proteolysis-dependent receptors

The Alzheimer’s amyloid precursor protein (APP) belongs to a conserved gene family that also includes the mammalian APLP1 and APLP2, the Drosophila APPL, and the C. elegans APL-1. The biological function of APP is still not fully clear. However, it is known that the APP family proteins have redundant and partly overlapping functions, which demonstrates the importance of studying all APP family members to gain a more complete picture. When APP was first cloned, it was speculated that it could function as a receptor. This theory has been further substantiated by studies showing that APP and its homologues bind both extracellular ligands and intracellular adaptor proteins. The APP family proteins undergo regulated intramembrane proteolysis (RIP), generating secreted and cytoplasmic fragments that have been ascribed different functions. In this review, we will discuss the APP family with focus on biological functions, binding partners, and regulated processing.     

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 nerve growth factor-like polypeptides and immunoreactivity related to the two types of neurotrophin receptors in earthworm tissues

Nerve growth factor (NGF) belongs by sequence homology to the neurotrophins, a family of proteins binding the same p75 receptor and closely related members of the Trk family of receptor tyrosine kinases. Fundamental in the vertebrate nervous system, neurotrophin signals have also been suggested as essential for relatively complex nervous systems occurring in invertebrate species that live longer than Caenorhabditis elegans and Drosophila melanogaster. Mammalian neurotrophins have been found to influence invertebrate neuronal growth. However, there are only a few data on the presence of molecules related to neurotrophin signalling components in invertebrates. Our studies provide evidence that analogues of neurotrophins and neurotrophin receptors are expressed in Eisenia foetida earthworms. In particular, NGF-like and Trk-like immunoreactive proteins are both expressed in the nervous system, whereas p75-like positivity identifies tubular structures associated with dorsal pores that are involved in the earthworm response to mechanical irritation or stress. Received 12 November 2001; received after revision 8 January 2002; accepted 8 January 2002     

Signal regulation by family conspiracy

The signal regulating proteins (SIRPs) are a family of ubiquitously expressed transmembrane glycoproteins composed of two subgroups: SIRPα and SIRPβ, containing more than ten members. SIRPα has been shown to inhibit signalling through a variety of receptors including receptor tyrosine kinases and cytokine receptors. This function involves protein tyrosine kinases and is dependent on immunoreceptor tyrosine-based inhibition motifs which recruit key protein tyrosine phosphatases to the membrane. Negative regulation by SIRPα may also involve its ligand, CD47, in a bi-directional signalling mechanism. The SIRPβ subtype has no cytoplasmic domain but instead associates with at least one other transmembrane protein (DAP-12, or KARAP). DAP-12 possesses immunoreceptor tyrosine-based activation motifs within its cytoplasmic domain that are thought to link SIRPβ to activating machinery. SIRPα and SIRPβ thus have complementary roles in signal regulation and may conspire to tune the response to a stimulus. Received 6 July 2000; revised 2 August 2000; accepted 5 August 2000     

The Nudix hydrolase superfamily

Nudix hydrolases are found in all classes of organism and hydrolyse a wide range of organic pyrophosphates, including nucleoside di- and triphosphates, dinucleoside and diphosphoinositol polyphosphates, nucleotide sugars and RNA caps, with varying degrees of substrate specificity. Some superfamily members, such as Escherichia coli MutT, have the ability to degrade potentially mutagenic, oxidised nucleotides while others control the levels of metabolic intermediates and signalling compounds. In prokaryotes and simple eukaryo tes, the number of Nudix genes varies from 0 to over 30, reflecting the metabolic complexity and adaptability of the organism. Mammals have around 24 Nudix genes, several of which encode more than one variant. This review integrates the sizeable recent literature on these proteins with information from global functional genomic studies to provide some insights into the possible roles of different superfamily members in cellular metabolism and homeostasis and to stimulate discussion and further research into this ubiquitous protein family. Received 16 August 2005; received after revision 3 October 2005; accepted 13 October 2005     

Diversity and specificity of the mitogen-activated protein kinase phosphatase-1 functions

The balance of protein phosphorylation is achieved through the actions of a family of protein serine/threonine kinases called the mitogen-activated protein kinases (MAPKs). The propagation of MAPK signals is attenuated through the actions of the MAPK phosphatases (MKPs). The MKPs specifically inactivate the MAPKs by direct dephosphorylation. The archetypal MKP family member, MKP-1 has garnered much of the attention amongst its ten other MKP family members. Initially viewed to play a redundant role in the control of MAPK signaling, it is now clear that MKP-1 exerts profound regulatory functions on the immune, metabolic, musculoskeletal and nervous systems. This review focuses on the physiological functions of MKP-1 that have been revealed using mouse genetic approaches. The implications from studies using MKP-1-deficient mice to uncover the role of MKP-1 in disease will be discussed.     

The <Emphasis Type="Italic">Adhesion</Emphasis> GPCRs: A unique family of G protein-coupled receptors with important roles in both central and peripheral tissues

G protein-coupled receptors (GPCRs) are a diverse superfamily of membrane-bound receptors. The second largest subgroup of GPCRs, the Adhesion GPCRs, has 33 members in humans. Phylogenetic analysis of the entire repertoire of the seven transmembrane- domain (7TM) regions of GPCRs shows that the Adhesion GPCRs form a distinct family. Adhesion GPCRs are characterised by (1) long N termini with multiple functional domains often found in other proteins such as tyrosine kinases, integrins and cadherins, (2) highly complex genomic structure with multiple introns and splice variants and (3) a 7TM region that has no clear similarities with 7TM from other GPCRs. Several Adhesion GPCRs are known to have a role in the immune system but it is becoming more evident that many have important roles in the CNS. We speculate that the overall structural construction of the Adhesion GPCRs allows them to participate in different types of cell guidance. Received 8 February 2007; received after revision 21 March 2007; accepted 25 April 2007     

Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria

The finding that mitochondria contain substrates for protein kinases lead to the discovery that protein kinases are located in the mitochondria of certain tissues and species. These include pyruvate dyhydrogenase kinase, branched-chain α-ketoacid dehydrogenase kinase, protein kinase A, protein kinase Cδ, stress-activated kinase and A-Raf as well as unidentified kinases. Recent evidence suggests that mitochondrial protein kinases may be involved in physiological processes such as apoptosis and steroidogenesis. Additionally, the novel finding of low-molecular-weight GTP-binding proteins in mitochondria suggests the possibility that these may interact with mitochondrial protein kinases to regulate the activity of mitochondrial effector proteins. The fact that there are components of cellular regulatory systems in mitochondria indicates the exciting possibility of undiscovered systems regulating mitochondrial physiology. Received 19 June 2001; received after revision 7 August 2001; accepted 8 August 2001     

Role of AGC kinases in plant growth and stress responses

AGC kinases are important regulators of cell growth, metabolism, division, and survival in mammalian systems. Mutation or deregulation of members of this family of protein kinases contribute to the pathogenesis of many human diseases, including cancer and diabetes. Although AGC kinases are conserved in the plant kingdom, little is known about their molecular functions and targets. Some of the best-studied plant AGC kinases mediate auxin signaling and are thereby involved in the regulation of growth and morphogenesis. Furthermore, certain members are regulated by lipid-derived signals via the 3-phosphoinositide-dependent kinase 1 (PDK1) and the kinase target of rapamycin (TOR), similar to its animal counterparts. In this review, we discuss recent findings on plant AGC kinases that unravel important roles in the regulation of plant growth, immunity and cell death, and connections to stress-induced mitogen-activated protein kinase signaling cascades.     

Structure and dynamic regulation of Src-family kinases

Src-family kinases are modular signaling proteins involved in a diverse array of cellular processes. All members of the Src family share the same domain organization, with modular SH3, SH2 and kinase domains followed by a C-terminal negative regulatory tail. X-ray crystallographic analyses of several Src family members have revealed critical roles for the SH3 and SH2 domains in the down-regulation of the kinase domain. This review focuses on biological, biophysical, and computational studies that reveal conformationally distinct active states within this unique kinase family. Received 10 March 2008; received after revision 17 May 2008; accepted 21 May 2008     

VRK1 and AURKB form a complex that cross inhibit their kinase activity and the phosphorylation of histone H3 in the progression of mitosis

Regulation of cell division requires the integration of signals implicated in chromatin reorganization and coordination of its sequential changes in mitosis. Vaccinia-related kinase 1 (VRK1) and Aurora B (AURKB) are two nuclear kinases involved in different steps of cell division. We have studied whether there is any functional connection between these two nuclear kinases, which phosphorylate histone H3 in Thr3 and Ser10, respectively. VRK1 and AURKB are able to form a stable protein complex, which represents only a minor subpopulation of each kinase within the cell and is detected following nocodazole release. Each kinase is able to inhibit the kinase activity of the other kinase, as well as inhibit their specific phosphorylation of histone H3. In locations where the two kinases interact, there is a different pattern of histone modifications, indicating that there is a local difference in chromatin during mitosis because of the local complexes formed by these kinases and their asymmetric intracellular distribution. Depletion of VRK1 downregulates the gene expression of BIRC5 (survivin) that recognizes H3-T3ph, both are dependent on the activity of VRK1, and is recovered with kinase active murine VRK1, but not with a kinase-dead protein. The H3–Thr3ph–survivin complex is required for AURB recruitment, and their loss prevents the localization of ACA and AURKB in centromeres. The cross inhibition of the kinases at the end of mitosis might facilitate the formation of daughter cells. A sequential role for VRK1, AURKB, and haspin in the progression of mitosis is proposed.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> innate immunity: an emerging role for peptidoglycan recognition proteins in bacteria detection

Over the past years, parallel studies conducted in mammals and flies have emphasized the existence of common mechanisms regulating the vertebrate and invertebrate innate immune systems. This culminated in the discovery of the central role of the Toll pathway in Drosophila immunity and in the implication of Toll-like receptors (TLRs)/interleukin-1(IL-1) in the mammalian innate immune response. In spite of clear similarities, such as shared intracellular pathway components, important divergences are expected between the two groups, whose last common ancestor lived more than half a billion years ago. The most obvious discrepancies lie in the mode of activation of the signalling receptors by microorganisms. In mammals, TLRs are part of protein complexes which directly recognize microbe-associated patterns, whereas Drosophila Toll functions like a classical cytokine receptor rather than a pattern recognition receptor. Recent studies demonstrate that members of the evolutionarily conserved peptidoglycan recognition protein family play an essential role in microbial sensing during immune response of Drosophila.Received 26 June 2003; received after revision 29 July 2003; accepted 25 August 2003     

Cyanobacterial psbA gene family: optimization of oxygenic photosynthesis

The D1 protein of Photosystem II (PSII), encoded by the psbA genes, is an indispensable component of oxygenic photosynthesis. Due to strongly oxidative chemistry of PSII water splitting, the D1 protein is prone to constant photodamage requiring its replacement, whereas most of the other PSII subunits remain ordinarily undamaged. In cyanobacteria, the D1 protein is encoded by a psbA gene family, whose members are differentially expressed according to environmental cues. Here, the regulation of the psbA gene expression is first discussed with emphasis on the model organisms Synechococcus sp. and Synechocystis sp. Then, a general classification of cyanobacterial D1 isoforms in various cyanobacterial species into D1_m, D1:1, D1:2, and D1′ forms depending on their expression pattern under acclimated growth conditions and upon stress is discussed, taking into consideration the phototolerance of different D1 forms and the expression conditions of respective members of the psbA gene family.     

The Janus kinase family of protein tyrosine kinases and their role in signaling

In the early 1990s, the search for protein kinases led to the discovery of a novel family of non-receptor tyrosine kinases, the Janus kinases or JAKs. These proteins were unusual because they contained two kinase homology domains and no other known signaling modules. It soon became clear that these were not ‘just another’ type of kinase. Their ability to complement mutant cells insensitive to interferons and to be activated by a variety of cytokines demonstrated their central signaling function. Now, as we approach the end of the decade, it is evident from biochemical studies to knockout mice that JAKs play non-redundant functions in development, differentiation, and host defense mechanisms. Here, recent progress is reviewed, with particular emphasis on structure-function studies aimed at revealing how this family of tyrosine kinases is regulated.