Human cdc2 protein kinase is a major cell-cycle regulated tyrosine kinase substrate

cdc2 is a catalytic subunit of a protein kinase complex, called the M-phase promoting factor, that induces entry into mitosis and is universal among eukaryotes. In HeLa cells, cdc2 is shown to be the most abundant phosphotyrosine-containing protein and its phosphotyrosine content is subject to cell-cycle regulation. One site of cdc2 tyrosine phosphorylation in vivo is selectively phosphorylated by pp60c-src in vitro.     

Human transforming growth factors induce tyrosine phosphorylation of EGF receptors

Cultured cell lines of human tumour origin as well as cells transformed by various RNA tumour viruses secrete low molecular weight polypeptide transforming growth factors (TGFs). In addition to competing with epidermal growth factor (EGF) for binding to its cellular receptor, TGFs can transform morphologically fibroblast and epithelial cells in culture. In view of accumulating evidence that tyrosine phosphorylation activity is associated with the transforming genes of various tumour viruses, we determined whether phosphotyrosine levels were elevated in these human tumour cells. We show here that TGFs produced by human tumour cells induce phosphorylation of specific tyrosine acceptor sites in the 160,000-molecular weight (160 K) EGF receptor.     

Phorbol ester and diacylglycerol induce protein phosphorylation at tyrosine

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is an efficient tumour promoter in vivo. In vitro, TPA activates the phospholipid- and Ca2+-dependent protein kinase, kinase C. This activation is believed to reflect the structural similarity between TPA and diacylglycerol, the endogenous protein kinase C activator which is produced in vivo by hydrolysis of phosphatidylinositol (reviewed in ref. 3). Protein kinase C phosphorylates protein substrates at serine and threonine residues in vitro. The effects of TPA on cultured fibroblasts--including enhanced hexose uptake, disruption of actin stress fibres and growth stimulation--are very similar to those induced by certain retrovirus transforming proteins and by peptide growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and multiplication-stimulating activity (MSA). These transforming proteins and mitogenic agents seem to act by inducing tyrosine-specific protein phosphorylation. Such observations suggested that some of the effects of TPA in vivo may be mediated by protein phosphorylation at tyrosine residues. A 42,000-molecular weight (42 K) polypeptide was previously shown to be phosphorylated at tyrosine in cells transformed by avian sarcoma viruses and in cells stimulated by EGF, PDGF or MSA (J. Cooper, personal communication and refs 11 and 12; this polypeptide was originally designated 43 K or spot n in ref. 10). We show here that this polypeptide also becomes phosphorylated at tyrosine in cells treated with TPA. Furthermore, exogenously added diacylglycerol likewise stimulates the phosphorylation of this protein at tyrosine.     

Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis.

Stimulation of growth factor receptors with tyrosine kinase activity is followed by rapid receptor dimerization, tyrosine autophosphorylation and phosphorylation of signalling molecules such as phospholipase C gamma (PLC gamma) and the ras GTPase-activating protein. PLC gamma and GTPase-activating protein bind to specific tyrosine-phosphorylated regions in growth factor receptors through their src-homologous SH2 domains. Growth factor-induced tyrosine phosphorylation of PLC gamma is essential for stimulation of phosphatidylinositol hydrolysis in vitro and in vivo. We have shown that a short phosphorylated peptide containing tyrosine at position 766 from a conserved region of the fibroblast growth factor (FGF) receptor is a binding site for the SH2 domain of PLC gamma (ref. 8). Here we show that an FGF receptor point mutant in which Tyr 766 is replaced by a phenylalanine residue (Y766F) is unable to associate with and tyrosine-phosphorylate PLC gamma or to stimulate hydrolysis of phosphatidylinositol. Nevertheless, the Y766F FGF receptor mutant can be autophosphorylated, and can phosphorylate several cellular proteins and stimulate DNA synthesis. Our data show that phosphorylation of the conserved Tyr 766 of the FGF receptor is essential for phosphorylation of PLC gamma and for hydrolysis of phosphatidylinositol, but that elimination of this hydrolysis does not affect FGF-induced mitogenesis.     

Cytoskeleton-associated protein tyrosine phosphorylation involved in induction of differentiation in mouse melanoma cells

The malignancy of a cancer is due partly to its poor differentiation. Genistein, a protein tyrosine kinase inhibitor, is found to induce the highly malignant B16-BL6 mouse melanoma cells to differentiate to mature phenotypes. When Triton X-100 insoluble fraction of the differentiated cells is prepared and analyzed, tyrosine phosphorylation levels of three cytoskeleton-associated proteins (65, 60 and 53 ku respectively) are found to decrease dramatically. But no any change is found when phosphotyrosine contents of the cytosol fraction or the total cellular protein preparations are evaluated. It is concluded that cytoskeleton-associated protein tyrosine phosphorylation may be involved in the control of differentiation of cancer cells. The decrease of phosphotyrosine contents of cytoskeleton-associated proteins may be one of the important mechanisms underlying the differentiation induction of cancer cells by anticancer agents.     

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.     

Increased phosphorylation of ribosomal protein S6 following microinjection of insulin receptor-kinase into Xenopus oocytes

The protein products of several transforming retroviruses as well as the receptors for several hormones and growth factors, including insulin, have been shown to possess a protein kinase activity in vitro specific for tyrosine residues in protein substrates, including themselves. In the case of pp60src and the insulin receptor, autophosphorylation activates the tyrosine kinase activity towards exogenous substrates. Experiments indicate that, in vivo, many of these viruses or growth factors induce an increase in cellular phosphotyrosine, as well as an increase in the phosphorylation of serine residues on proteins, including ribosomal protein S6. It seems likely that some of the effects of insulin might be mediated by phosphorylation of intracellular substrates by its receptor. As the beta subunit of the receptor is a transmembrane protein, such phosphorylation could occur either while the receptor is still in the membrane or after its internalization. In various cell systems, internalized receptors are degraded, reshuttled back to the plasmalemma or maintained in a separate compartment before reinsertion in the membrane; shuttling of the insulin receptor could provide the opportunity for it to phosphorylate various intracellular components as part of its mechanism of signal transduction. To approach directly the question of whether the receptor can elicit a signal while acting at an intracellular location, we have microinjected Xenopus oocytes with the insulin receptor kinase. The results indicate that an S6 protein-serine kinase is stimulated or an S6 protein-serine phosphatase inhibited by the activity of the insulin receptor, supporting the concept that the insulin receptor acting within the cell can elicit a biological response.     

A Tyr/Ser protein phosphatase encoded by vaccinia virus.

Protein tyrosine phosphorylation is associated with alterations in receptor activity, cellular proliferation and modulation of the cell cycle. Inappropriate tyrosine phosphorylation can lead to unrestrained cell growth and oncogenesis. Enzymes important in tyrosine dephosphorylation have also been described. Protein tyrosine phosphatases (PTPases) consist of two families. There is a receptor-like family of PTPases with an extracellular domain, transmembrane-spanning region and typically two repeated phosphatase domains. Proteins of the non-receptor-like family have a single catalytic phosphatase domain, show a substrate specificity for Tyr phosphate and will not hydrolyse Ser or Thr phosphate. Here we report that the vaccinia virus genome contains an open reading frame which shares amino-acid sequence identity with the PTPases. The purified protein encoded by the vaccinia virus H1 open reading frame expressed in bacteria hydrolyses substrates containing phosphotyrosine and phosphoserine. Mutagenesis of an essential Cys in the vaccinia phosphatase abolishes catalytic activity directed towards both substrates, suggesting that hydrolysis proceeds by a common mechanism. Understanding the function of the H1-encoded protein will help to define the role of the phosphatase in viral replication and pathogenesis.     

Regulation of repulsion versus adhesion by different splice forms of an Eph receptor

Eph tyrosine kinase receptors and their membrane-bound ephrin ligands mediate cell interactions and participate in several developmental processes. Ligand binding to an Eph receptor results in tyrosine phosphorylation of the kinase domain, and repulsion of axonal growth cones and migrating cells. Here we report that a subpopulation of ephrin-A5 null mice display neural tube defects resembling anencephaly in man. This is caused by the failure of the neural folds to fuse in the dorsal midline, suggesting that ephrin-A5, in addition to its involvement in cell repulsion, can participate in cell adhesion. During neurulation, ephrin-A5 is co-expressed with its cognate receptor EphA7 in cells at the edges of the dorsal neural folds. Three different EphA7 splice variants, a full-length form and two truncated versions lacking kinase domains, are expressed in the neural folds. Co-expression of an endogenously expressed truncated form of EphA7 suppresses tyrosine phosphorylation of the full-length EphA7 receptor and shifts the cellular response from repulsion to adhesion in vitro. We conclude that alternative usage of different splice forms of a tyrosine kinase receptor can mediate cellular adhesion or repulsion during embryonic development.     

Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts

Fibroblastic cultures derived from normal human tissues undergo a finite number of population doublings when serially subcultivated in vitro (see refs 1, 2 for reviews). Epidermal growth factor (EGF) serves as a mitogen for early doubling level cultures of the human fetal lung-derived cell strain, WI-38, under serum-free conditions. The ability of cells from late doubling level cultures to respond mitogenically to EGF is lost, however, despite undiminished binding of EGF throughout the replicative lifespan. The ultimate effects of EGF, that is DNA synthesis and mitosis (see ref. 4 for review), occur after a sequence of events initiated by binding of ligand to specific cellular receptors. The receptor for EGF has been characterized as a 145,000-165,000 (145 K-165 K) molecular weight doublet, and, like the receptors for platelet-derived growth factor and insulin, and the transforming proteins of certain of the RNA tumour viruses, is a tyrosine-specific protein kinase with autophosphorylating activity. Moreover, several of the cellular target molecules of tyrosine phosphorylation have been found to be substrates for two or more of these kinases. The hypothesis that tyrosine phosphorylation underlies a common mechanism of growth control prompted us to ask whether the loss of responsiveness to EGF by late doubling level WI-38 cells is accompanied by altered expression of the EGF receptor, and specifically whether changes occur in the ability of receptors from populations of cells of various in vitro ages to catalyse tyrosine autophosphorylation. We show here that autophosphorylating activity is absent from the EGF receptor of cells which have lost their mitogenic responsiveness to EGF.     

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.     

Altered tyrosine 527 phosphorylation and mitotic activation of p60c-src.

The tyrosine kinasee activity of p60c-src, the protein product of the c-src gene, increases during mitosis; this may be important in initiating at least some of the cellular changes that occur during this phase of the cell cycle. Although there is evidence that p60c-src is phosphorylated at several sites during mitosis, phosphorylation in vitro does not increase its kinase activity. We now report that the kinase activity of a p60c-src mutant with residue tyrosine 527 changed to phenylanine does not change during the cell cycle, suggesting that changes in the phosphorylation state of this residue may be responsible for the activation of p60c-src at mitosis. Although changes in phosphorylation at Tyr 527 cannot be detected with the wild-type protein we find that phosphorylation at Tyr 527 of a mutant with reduced kinase activity decreases threefold during mitosis. On the basis of these results we suggest that activation of p60c-src at mitosis results from decreased phosphorylation on Tyr 527, and that p60c-src may be or may activate the kinase that phosphorylates Tyr 527.     

Autophosphorylation sites on the epidermal growth factor receptor

The epidermal growth factor (EGF) receptor is a tyrosine-specific protein kinase with autophosphorylating activity. A 300 amino acid-long region of the receptor's cytoplasmic domain matches (35-90% homology) sequences of transforming proteins from the src family and includes a putative nucleotide binding site. Several of the src transforming proteins have tyrosine kinase activity, but v-erb-B, which appears to be a truncated EGF receptor, is virtually identical to the receptor over this region and yet lacks detectable kinase activity. To locate possible acceptor sites in the v-erb-B protein, we have mapped these sites in the human EGF receptor. We report here that three tyrosine sites near the C-terminus are phosphorylated in vitro. In intact cells, we find that EGF stimulates phosphorylation of several sites, the tyrosine 14 residues from the C-terminus being modified the most extensively. The equivalent site is absent in the v-erb-B protein of avian erythroblastosis virus (AEV) and may influence tyrosine kinase activity.     

Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases.

Mitogen activated protein (MAP) kinases (MAPKs) are a family of protein-serine/threonine kinases activated as an early intracellular response to a variety of hormones and growth factors. They are unique in requiring both serine/threonine and tyrosine phosphorylation to become active and are the only examples of protein-serine/threonine kinases activated by tyrosine phosphorylation. Nerve growth factor (NGF) promotes differentiation of phaeochromocytoma (PC12) cells, which respond by conversion within hours from a chromaffin-like to a sympathetic neuron-like phenotype. NGF stimulation of PC12 cells increases the activity of two protein kinases by greater than 20-fold within minutes, both strikingly similar to MAPKs. They are inactivated by either protein-tyrosine phosphatases or the protein-serine/threonine phosphatase termed protein phosphatase 2A (ref. 8), they activate protein S6 kinase-II (refs 9, 10), and they phosphorylate identical threonine residues on myelin basic protein (our unpublished results) to those phosphorylated by other MAPKs. Immunological data indicate that these protein kinases, termed peak-I and peak-II (Fig. 1a) are probably ERK2 and ERK1, respectively, two widely expressed MAPK isoforms. Here we identify the 'MAP kinase kinases' (MAPKKs) in PC12 cells which are activated by NGF and report that MAPKKs are dependent on serine/threonine phosphorylation for activity and promote phosphorylation of serine/threonine and tyrosine residues on MAPKs.     

Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis.

Stimulation of certain receptor tyrosine kinases results in the tyrosine phosphorylation and activation of phospholipase C gamma (PLC gamma), an enzyme that catalyses the hydrolysis of phosphatidylinositol (PtdIns). This hydrolysis generates diacylglycerol and free inositol phosphate, which in turn activate protein kinase C and increase intracellular Ca2+, respectively. PLC gamma physically associates with activated receptor tyrosine kinases, suggesting that it is a substrate for direct phosphorylation by these kinases. Here we report that a fibroblast growth factor (FGF) receptor with a single point mutation at residue 766 replacing tyrosine with phenylalanine fails to associate with PLC gamma in response to FGF. This mutant receptor also failed to mediate PtdIns hydrolysis and Ca2+ mobilization after FGF stimulation. However, the mutant receptor phosphorylated itself and several other cellular proteins, and it mediated mitogenesis in response to FGF. These findings show that a point mutation in the FGF receptor selectively eliminates activation of PLC gamma and that neither Ca2+ mobilization nor PtdIns hydrolysis are required for FGF-induced mitogenesis.     

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.     

Polyoma virus transforming protein associates with the product of the c-src cellular gene

Polyoma virus can transform the growth properties of rodent cells grown in culture and form tumours in susceptible animals, an activity largely due to one of the virus-encoded proteins, called middle T. Middle T has an associated tyrosine-specific protein kinase activity in vitro and interacts with cellular membranes, but the biochemical basis of its ability to transform remains unclear. Although there is some correlation between the transforming activity of different polyoma virus mutants and their ability to accept phosphate on tyrosine in middle T in the in vitro kinase reaction, the abundance of phosphotyrosine in protein is not elevated in polyoma virus-transformed cells and no cellular substrates for the putative kinase have been identified. It is also not yet known whether the tyrosine kinase of middle T is an intrinsic activity of the protein itself or the property of an associated enzyme. The experiments described here indicate that a fraction of middle T forms a stable complex with pp60c-src, the product of a cellular oncogene, and lead us to propose that the middle T associated kinase at least in part is a property of pp60c-src rather than middle T itself.