Genome-wide survey of protein kinases required for cell cycle progression

Cycles of protein phosphorylation are fundamental in regulating the progression of the eukaryotic cell through its division cycle. Here we test the complement of Drosophila protein kinases (kinome) for cell cycle functions after gene silencing by RNA-mediated interference. We observed cell cycle dysfunction upon downregulation of 80 out of 228 protein kinases, including most kinases that are known to regulate the division cycle. We find new enzymes with cell cycle functions; some of these have family members already known to phosphorylate microtubules, actin or their associated proteins. Additionally, depletion of several signalling kinases leads to specific mitotic aberrations, suggesting novel roles for familiar enzymes. The survey reveals the inter-digitation of systems that monitor cellular physiology, cell size, cellular stress and signalling processes with the basic cell cycle regulatory machinery.     

Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation.

Increasing evidence indicates that the integrin family of cell adhesion receptors can transduce biochemical signals from the extracellular matrix to the cell interior to modulate cell growth and differentiation. We have shown that integrin/ligand interactions can trigger tyrosine phosphorylation of a protein of M(r) 120,000 (pp120), so it is possible that signal transduction by integrins might involve activation of intracellular protein tyrosine kinases as an early event in cell binding to the extracellular matrix. Here we report that pp120 is identical to the focal adhesion-associated protein tyrosine kinase pp125FAK (refs 3, 4). We show that tyrosine phosphorylation of this protein is modulated both by cell adhesion and transformation by pp60v-src, and that these changes in phosphorylation are correlated with increased pp125FAK tyrosine kinase activity. A model is proposed to relate these findings to the molecular basis of anchorage-independent growth of transformed cells.     

Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain

Tyrosine-specific phosphorylation of proteins is a key to the control of diverse pathways leading to cell growth and differentiation. The protein-tyrosine kinases described to date are either transmembrane proteins having an extracellular ligand binding domain or cytoplasmic proteins related to the v-src oncogene. Most of these proteins are expressed in a wide variety of cells and tissues; few are tissue-specific. Previous studies have suggested that lymphokines could mediate haematopoietic cell survival through their action on glucose transport, regulated in some cells through the protein-tyrosine kinase activity of the insulin receptor. We have investigated the possibility that insulin receptor-like genes are expressed specifically in haematopoietic cells. Using the insulin receptor-related avian sarcoma oncogene v-ros as a probe, we have isolated and characterized the complementary DNA of a novel gene, ltk (leukocyte tyrosine kinase). The ltk gene is expressed mainly in leukocytes, is related to several tyrosine kinase receptor genes of the insulin receptor family and has unique structural properties: it apparently encodes a transmembrane protein devoid of an extracellular domain. Two candidate ltk proteins have been identified with antibodies in the mouse thymus, and have properties indicating that they are integral membrane proteins. These features suggest that ltk could be a signal transduction subunit for one or several of the haematopoietic receptors.     

Functional modulation of the nicotinic acetylcholine receptor by tyrosine phosphorylation

Tyrosine-specific protein phosphorylation has been implicated in the regulation of cell transformation and proliferation. However, recent studies have shown that the expression of protein tyrosine kinases in adult brain is very high, suggesting that tyrosine-specific protein phosphorylation may also have a role in the regulation of neuronal function. Although a number of substrate proteins are phosphorylated on tyrosine residues, the functional alteration of proteins by tyrosine phosphorylation has previously been convincingly demonstrated only for protein tyrosine kinases. The nicotinic acetylcholine receptor, a neurotransmitter-gated ion channel, is phosphorylated by a protein tyrosine kinase in post-synaptic membranes in vitro and in vivo. We demonstrate here that this tyrosine phosphorylation increases the rate of the rapid phase of desensitization of the nicotinic receptor, as measured by single channel recording of purified nicotinic acetylcholine receptor, when reconstituted in lipid vesicles. These data provide direct evidence for the regulation of ion channel properties by tyrosine phosphorylation. The results, which demonstrate a functional role of tyrosine phosphorylation in the nervous system, suggest a widespread role for tyrosine phosphorylation in neuronal signal transduction.     

Epidermal growth factor-dependent phosphorylation of lipocortin

Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein.     

Spred is a Sprouty-related suppressor of Ras signalling

Cellular proliferation, and differentiation of cells in response to extracellular signals, are controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Here we describe two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signalling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibited growth-factor-mediated activation of MAP kinase. The Ras-MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection revealed that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associated with Ras but did not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibited the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras-Raf interaction and MAP kinase signalling.     

Calcium-dependent phosphorylation of histone H3 in butyrate-treated HeLa cells

Ca2+ is prominant in the control of cell proliferation and function. However, the biochemical mechanism(s) mediating its effects on nuclear events is unknown. We report here that Ca2+, at physiological concentrations, stimulates the phosphorylation of histone H3 by an endogenous protein kinase in HeLa cell nuclei. Also, pretreatment of cells with Na butyrate, which increases histone acetylation, selectively increases the susceptability of histone H3 to phosphorylation by the protein kinase. Our results reveal a potential link between histone H3 acetylation and phosphorylation, modifications which are thought to have important effects on chromatin structure and function and suggest a possible mechanism whereby stimuli at the cell surface (such as hormones, mitogens and drugs) may influence biochemical events at the nuclear level; changes in the intracellular Ca2+ concentration may influence the phosphorylation of chromosomal proteins, mediated by Ca2+ -dependent kinases in th nucleus.     

Roles of MAP kinase signaling pathway in oocyte meiosis

Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases expressed widely in eukaryotic cells. MAPK is activated by a cascade of protein kinase phosphorylation and plays pivotal roles in regulating meiosis process in oocytes. As an important physical substrate of MAPK, p90^rsk mediates numerous MAPK functions. MAPK was activated at G2/M transition during meiosis. Its activity reached the peak at MⅠ stage and maintained at this level until the time before the pronuclear formation after fertilization. There is complex interplay between MAPK and MPF in the meiosis regulation. Furthermore, other intracellular signal transducers, such as cAMP, protein kinase C and protein phosphotase, ect., also regulated the activity of MAPK at different stages during meiosis in oocytes. In the present article, the roles of MAPK signaling pathway in oocyte meiosis are reviewed and discussed.     

Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine

The protein responsible for malignant transformation by avian sarcoma viruses (ASVs) has been identified as a phosphoprotein of molecular weight 60,000 designated pp60src (refs 1--4). It has been suggested that this protein has a functional role in cellular transformation involving the phosphorylation of cellular proteins, for it was discovered that specific immunoprecipitates from ASV-transformed cells that contain pp60src catalysed the transfer of phosphate from [gamma-32P]ATP to the heavy chain of rabbit immunoglobulin. Additional studies involving the cell-free synthesis of the ASV src protein further demonstrated that the presence of the src polypeptide correlated with that presence of a phosphotransferase activity. Our studies, involving the biochemical purification of this protein, have demonstrated that the ASV-transforming gene product, pp60src, is itself a protein kinase. We have purified the pp60src protein approximately 5,000-fold using either conventional ion-exchange chromatography or immunoaffinity chromatography. The resultant partially purified preparations contain a cyclic AMP-independent protein kinase activity. We report here that the soluble phosphotransferase activity of partially purified pp60src results in the phosphorylation of exclusively tyrosine residues in a variety of proteins that serve as substrates.     

Targets of the cyclin-dependent kinase Cdk1

The events of cell reproduction are governed by oscillations in the activities of cyclin-dependent kinases (Cdks). Cdks control the cell cycle by catalysing the transfer of phosphate from ATP to specific protein substrates. Despite their importance in cell-cycle control, few Cdk substrates have been identified. Here, we screened a budding yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts. We identified about 200 Cdk1 substrates, several of which are phosphorylated in vivo in a Cdk1-dependent manner. The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events. Detailed analysis of these substrates is likely to yield important insights into cell-cycle regulation.     

Abolition of actin-bundling by phosphorylation of human erythrocyte protein 4.9

Protein 4.9, first identified as a component of the human erythrocyte membrane skeleton, binds to and bundles actin filaments. Protein 4.9 is a substrate for various kinases, including a cyclic AMP(cAMP)-dependent one, in vivo and in vitro. We show here that phosphorylation of protein 4.9 by the catalytic subunit of cAMP-dependent protein kinase reversibly abolishes its actin-bundling activity, but phosphorylation by protein kinase C has no such effect. A quantitative immunoassay showed that human erythrocytes contain 43,000 trimers of protein 4.9 per cell, which is equivalent to one trimer for each actin oligomer in these red blood cells. As analogues of protein 4.9 have been identified together with analogues of other erythroid skeletal proteins in non-erythroid tissues of numerous vertebrates, phosphorylation and dephosphorylation of protein 4.9 may be the basis for a mechanism that regulates actin bundling in many cells.     

Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells

Phosphotyrosine-containing proteins are minor components of normal cells which appear to be associated primarily with the regulation of cellular metabolism and growth. The insulin receptor is a tyrosine-specific protein kinase, and one of the earliest detectable responses to insulin binding is activation of this kinase and autophosphorylation of its beta-subunit. Tyrosine autophosphorylation activates the phosphotransferase in the beta-subunit and increases its reactivity toward tyrosine phosphorylation of other substrates. When incubated in vitro with [gamma-32P]ATP and insulin, the purified insulin receptor phosphorylates various proteins on their tyrosine residues. However, so far no proteins other than the insulin receptor have been identified as undergoing tyrosine phosphorylation in response to insulin in an intact cell. Here, using anti-phosphotyrosine antibodies, we have identified a novel phosphotyrosine-containing protein of relative molecular mass (Mr) 185,000 (pp185) which appears during the initial response of hepatoma cells to insulin binding. In contrast to the insulin receptor, pp185 does not adhere to wheat-germ agglutininagarose or bind to anti-insulin receptor antibodies. Phosphorylation of pp185 is maximal within seconds after exposure of the cells to insulin and exhibits a dose-response curve similar to that of receptor autophosphorylation, suggesting that this protein represents the endogenous substrate for the insulin receptor kinase.     

Protein phosphorylation in the brain

Protein phosphorylation represents an approach, sometimes the only approach available, to study the molecular basis for a wide variety of neurophysiological phenomena. The injection of protein kinases or protein kinase inhibitors into neurones has provided direct evidence that activation of protein kinases has an obligatory role in the mechanisms by which numerous extracellular signals produce specific physiological responses in neurones. A diversity of substrate proteins for the kinases have already been found. In several instances, the identity and functional role of these substrate proteins have been established.     

FMRFamide reverses protein phosphorylation produced by 5-HT and cAMP in Aplysia sensory neurons

Neurotransmitter can modulate neuronal activity through a variety of second messengers that act on ion channels and other substrate proteins. The most commonly described effector mechanism for second messengers in neurons depends on protein phosphorylation mediated by one of three sets of kinases: the cyclic AMP-dependent protein kinases, the Ca2+-calmodulin-dependent protein kinases, and the Ca2+-phospholipid-dependent protein kinases. In addition, some neurotransmitters and second messengers can also inhibit protein phosphorylation by lowering cAMP levels (either by inhibiting adenylyl cyclase or activating phosphodiesterases). This raises the question: can neurotransmitters also modulate neuronal activity by decreasing protein phosphorylation that is independent of cAMP? Various biochemical experiments show that a decrease in protein phosphorylation can arise through activation of a phosphatase or inhibition of kinases. In none of these cases, however, is the physiological role for the decrease in protein phosphorylation known. Here we report that in Aplysia sensory neurons, the presynaptic inhibitory transmitter FMRFamide decreases the resting levels of protein phosphorylation without altering the level of cAMP. Furthermore, FMRFamide overrides the cAMP-mediated enhancement of transmitter release produced by 5-hydroxytryptamine (5-HT), and concomitantly reverses the cAMP-dependent increase in protein phosphorylation produced by 5-HT. These findings indicate that a receptor-mediated decrease in protein phosphorylation may play an important part in the modulation of neurotransmitter release.     

Synapsin I bundles F-actin in a phosphorylation-dependent manner

Synapsin I is a neuron-specific phosphoprotein localized to the cytoplasmic surface of synaptic vesicles. This phosphoprotein is a major substrate for cyclic AMP-dependent and calcium/calmodulin-dependent protein kinases. Its state of phosphorylation can be altered both in vivo and in vitro by a variety of physiological and pharmacological manipulations known to affect synaptic function. Recent direct evidence suggests that it may be involved in the regulation of neurotransmitter release from the nerve terminal. In the nerve terminal, synaptic vesicles are embedded in a cytoskeletal network, consisting in part of actin. We report here the ability of the dephospho-form of synapsin I to bundle F-actin. This bundling activity is reduced when synapsin I is phosphorylated by cAMP-dependent protein kinase and virtually abolished when it is phosphorylated by calcium/calmodulin-dependent protein kinase II or by both kinases. These results, demonstrating an interaction of synapsin I with actin in vitro, support the possibility that synapsin I is involved in clustering of synaptic vesicles at the presynaptic terminal and that the phosphorylation of synapsin I may be involved in regulating the translocation of synaptic vesicles to their sites of release.     

HeLa细胞中激酶NUAK1调控Tuberiun的磷酸化

NUAK1是LKB1的下游激酶之一,可被LKB1磷酸化而激活,但对其在LKB1相关信号通路中的功能仍缺乏了解.本研究发现在HeLa细胞中重建LKB1表达后NUAK1与tuberin蛋白免疫共沉淀,提示在野生型LKB1存在时NUAK1与tuberin可能存在直接相互作用.进一步的激酶活性测定和in vivo蛋白磷酸化实验表明:在HeLa细胞中葡萄糖匾乏条件下,野生型LKB1可显著激活NUAK1的激酶活性;而被激活的NUAK1可明显提高tuberin 的磷酸化水平,使用NUAK1 siRNA pool干扰NUAK1的表达则几乎将tuberin的磷酸化水平降低为零.上述结果表明NUAK1可能介导了LKB1对tuberin磷酸化的调节,进而下调mTOR通路,抑制蛋白质合成与细胞生长增殖.     

Neuropeptide Y functions as a neuroproliferative factor

Neuropeptide Y (NPY) has a number of functions in mammalian physiology. Here we identify a role for NPY in promoting proliferation of postnatal neuronal precursor cells. NPY is synthesized in the postnatal olfactory epithelium by sustentacular cells, previously proposed to function only in structural support. Mice with a targeted deletion of NPY contain half as many dividing olfactory neuronal precursor cells as do controls. Furthermore, NPY-deficient mice develop significantly fewer olfactory neurons by adulthood. NPY acts on multipotent neuronal precursor or basal cells to activate rapidly and transiently the extracellular signal-regulated kinase (ERK)1/2 subgroup of mitogen-activated protein kinases. The NPY Y1 receptor subtype appears to mediate this effect. The ability of NPY to induce neuronal precursor proliferation is mediated by protein kinase C (PKC), indicating an upstream PKC-dependent activation of ERK1/2. These results indicate that NPY may regulate neuronal precursor proliferation in the adult mammal.