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.     

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.     

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.     

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.     

Mechanistic principles of RAF kinase signaling

The RAF family of kinases are key components acting downstream of receptor tyrosine kinases and cells employ several distinct mechanisms to strictly control their activity. RAF transitions from an inactive state, where the N-terminal regulatory region binds intramolecularly to the C-terminal kinase domain, to an open state capable of executing the phosphoryl transfer reaction. This transition involves changes both within and between the protein domains in RAF. Many different proteins regulate the transition between inactive and active states of RAF, including RAS and KSR, which are arguably the two most prominent regulators of RAF function. Recent developments have added several new twists to our understanding of RAF regulation. Among others, dimerization of the RAF kinase domain is emerging as a crucial step in the RAF activation process. The multitude of regulatory protein–protein interactions involving RAF remains a largely untapped area for therapeutic applications.     

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.     

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.     

Mechanisms controlling cellular suicide: role of Bcl-2 and caspases

Apoptosis is an essential and highly conserved mode of cell death that is important for normal development, host defense and suppression of oncogenesis. Faulty regulation of apoptosis has been implicated in degenerative conditions, vascular diseases, AIDS and cancer. Among the numerous proteins and genes involved, members of the Bcl-2 family play a central role to inhibit or promote apoptosis. In this article, we present up-to-date information and recent discoveries regarding biochemical functions of Bcl-2 family proteins, positive and negative interactions between these proteins, and their modification and regulation by either proteolytic cleavage or by cytosolic kinases, such as Raf-1 and stress-activated protein kinases. We have critically reviewed the functional role of caspases and the consequences of cleaving key substrates, including lamins, poly(ADP ribose) polymerase and the Rb protein. In addition, we have presented the latest Fas-induced signalling mechanism as a model for receptor-linked caspase regulation. Finally, the structural and functional interactions of Ced-4 and its partial mam malian homologue, apoptosis protease activating factor-1 (Apaf-1), are presented in a model which includes other Apafs. This model culminates in a caspase/Apaf regulatory cascade to activate the executioners of programmed cell death following cytochrome c release from the mitochondria of mammalian cells. The importance of these pathways in the treatment of disease is highly dependent on further characterization of genes and other regulatory molecules in mammals. Received 18 February 1998; accepted February 1998     

Possible mechanisms and function of nuclear trafficking of the colony-stimulating factor-1 receptor

Receptor tyrosine kinases (RTK) have long being studied with respect to the “canonical” signaling. This includes ligand-induced activation of a receptor tyrosine kinase at the cell surface that leads to receptor dimerization, followed by its phosphorylation in the intracellular domain and activation. The activated receptor then recruits cytoplasmic signaling molecules including other kinases. Activation of the downstream signaling cascade frequently leads to changes in gene expression following nuclear translocation of downstream targets. However, RTK themselves may localize within the nucleus, as either full-length molecules or cleaved fragments, with or without their ligands. Significant differences in this mechanism have been reported depending on the individual RTK, cellular context or disease. Accumulating evidences indicate that the colony-stimulating factor-1 receptor (CSF-1R) may localize within the nucleus. To date, however, little is known about the mechanism of CSF-1R nuclear shuttling, as well as the functional role of nuclear CSF-1R.     

Positive regulation of apoptosis signal-regulating kinase 1 by dual-specificity phosphatase 13A

Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP kinase kinase kinase, is activated by several death stimuli and is tightly regulated by several mechanisms such as interactions with regulatory proteins and post-translational modifications. Here, we report that dual-specificity phosphatase 13A (DUSP13A) functions as a novel regulator of ASK1. DUSP13A interacts with the N-terminal domain of ASK1 and induces ASK1-mediated apoptosis through the activation of caspase-3. DUSP13A enhances ASK1 kinase activity and thus its downstream factors. Small interfering RNA (siRNA) analyses show that knock-down of DUSP13A in human neuroblastoma SK-N-SH cells reduces ASK1 kinase activity. The phosphatase activity of DUSP13A is not required for the regulation of ASK1. This regulatory action of DSUP13 on ASK1 activity involves competition with Akt1, a negative regulator of ASK1, for binding to ASK1. Taken together, this study provides novel insights into the role of DUSP13A in the precise regulation of ASK1.     

Novel actions of tyrphostin AG 879: inhibition of RAF-1 and HER-2 expression combined with strong antitumoral effects on breast cancer cells

Binding of growth factors to cell surface receptors activates protein tyrosine kinases (PTKs) that initiate cascades of downstream signaling events including the mitogen-activated protein (MAP) kinase cascade. This study reports that the PTK inhibitor AG 879 inhibits proliferation of human breast cancer cells through an effect involving inhibition of MAP kinase activation, but which cannot be explained by effects of AG 879 on its known PTK targets. Instead, AG 879 markedly inhibits expression of the RAF-1 gene, which encodes an upstream MAP kinase kinase kinase. Additionally, expression of HER-2, but not of other genes tested, is inhibited by this compound. These novel effects have to be considered when using AG 879 as a TRK-A and HER-2 inhibitor but may have useful therapeutic implications.     

The tyrosine phosphatase SHP-1 negatively regulates cytotrophoblast proliferation in first-trimester human placenta by modulating EGFR activation

Insulin-like growth factors (IGFs) influence placental cell (cytotrophoblast) kinetics. We recently reported that the protein tyrosine phosphatase (PTP) SHP-2 positively regulates IGF actions in the placenta. In other systems, the closely related PTP, SHP-1, functions as a negative regulator of signaling events but its role in the placenta is still unknown. We examined the hypothesis that SHP-1 negatively regulates IGF actions in the human placenta. Immunohistochemical (IHC) analysis demonstrated that SHP-1 is abundant in cytotrophoblast. SHP-1 expression was decreased in first-trimester placental explants using siRNA; knockdown did not alter IGF-induced proliferation but it significantly enhanced proliferation in serum-free conditions, revealing that placental growth is endogenously regulated. Candidate regulators were determined by using antibody arrays, Western blotting, and IHC to examine the activation status of multiple receptor tyrosine kinases (RTKs) in SHP-1-depleted explants; amongst the alterations observed was enhanced activation of EGFR, suggesting that SHP-1 may interact with EGFR to inhibit proliferation. The EGFR tyrosine kinase inhibitor PD153035 reversed the elevated proliferation seen in the absence of SHP-1. This study demonstrates a role for SHP-1 in human trophoblast turnover and establishes SHP-1 as a negative regulator of EGFR activation. Targeting placental SHP-1 expression may provide therapeutic benefits in common pregnancy conditions with abnormal trophoblast proliferation.     

<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.     

Domain structure and function of matrix metalloprotease 23 (MMP23): role in potassium channel trafficking

MMP23 is a member of the matrix metalloprotease family of zinc- and calcium-dependent endopeptidases, which are involved in a wide variety of cellular functions. Its catalytic domain displays a high degree of structural homology with those of other metalloproteases, but its atypical domain architecture suggests that it may possess unique functional properties. The N-terminal MMP23 pro-domain contains a type-II transmembrane domain that anchors the protein to the plasma membrane and lacks the cysteine-switch motif that is required to maintain other MMPs in a latent state during passage to the cell surface. Instead of the C-terminal hemopexin domain common to other MMPs, MMP23 contains a small toxin-like domain (TxD) and an immunoglobulin-like cell adhesion molecule (IgCAM) domain. The MMP23 pro-domain can trap Kv1.3 but not closely-related Kv1.2 channels in the endoplasmic reticulum, preventing their passage to the cell surface, while the TxD can bind to the channel pore and block the passage of potassium ions. The MMP23 C-terminal IgCAM domain displays some similarity to Ig-like C2-type domains found in IgCAMs of the immunoglobulin superfamily, which are known to mediate protein–protein and protein–lipid interactions. MMP23 and Kv1.3 are co-expressed in a variety of tissues and together are implicated in diseases including cancer and inflammatory disorders. Further studies are required to elucidate the mechanism of action of this unique member of the MMP family.     

The parvins

The parvins are a family of proteins involved in linking integrins and associated proteins with intracellular pathways that regulate actin cytoskeletal dynamics and cell survival. Both α-parvin (PARVA) and β-parvin (PARVB) localize to focal adhesions and function in cell adhesion, spreading, motility and survival through interactions with partners, such as integrin-linked kinase (ILK), paxillin, α-actinin and testicular kinase 1. A complex of PARVA with ILK and the LIM protein PINCH-1 is critical for cell survival in a variety of cells, including certain cancer cells, kidney podocytes and cardiac myocytes. While PARVA inhibits the activities of Rac1 and testicular kinase 1 and cell spreading, PARVB binds αPIX and α-actinin, and can promote cell spreading. In contrast to PARVA, PARVB inhibits ILK activity and reverses some of its oncogenic effects in cancer cells. This review focuses on the structure and function of the parvins and some possible roles in human diseases. Received 5 August 2005; received after revision 5 September 2005; accepted 22 September 2005     

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     

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