S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34cdc28.

In somatic cells, entry into mitosis depends on the completion of DNA synthesis. This dependency is established by S-phase feedback controls that arrest cell division when damaged or unreplicated DNA is present. In the fission yeast Schizosaccharomyces pombe, mutations that interfere with the phosphorylation of tyrosine 15 (Y15) of p34cdc2, the protein kinase subunit of maturation promoting factor, accelerate the entry into mitosis and abolish the ability of unreplicated DNA to arrest cells in G2. Because the tyrosine phosphorylation of p34cdc2 is conserved in S. pombe, Xenopus, chicken and human cells, the regulation of p34cdc2-Y15 phosphorylation could be a universal mechanism mediating the S-phase feedback control and regulating the initiation of mitosis. We have investigated these phenomena in the budding yeast Saccharomyces cerevisiae. We report here that the CDC28 gene product (the S. cerevisiae homologue of cdc2) is phosphorylated on the equivalent tyrosine (Y19) during S phase but that mutations that prevent tyrosine phosphorylation do not lead to premature mitosis and do not abolish feedback controls. We have therefore demonstrated a mechanism that does not involve tyrosine phosphorylation of p34 by which cells arrest their division in response to the presence of unreplicated or damaged DNA. We speculate that this mechanism may not involve the inactivation of p34 catalytic 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.     

Triggering of cyclin degradation in interphase extracts of amphibian eggs by cdc2 kinase

The cell cycles of early Xenopus embryos consist of a rapid succession of alternating S and M phases. These cycles are controlled by the activity of a protein kinase complex (cdc2 kinase) which contains two subunits. One subunit is encoded by the frog homologue of the fission yeast cdc2+ gene, p34cdc2 and the other is a cyclin. The concentration of cyclins follows a sawtooth oscillation because they accumulate in interphase and are destroyed abruptly during mitosis. The association of cyclin and p34cdc2 is not sufficient for activation of cdc2 kinase, however; dephosphorylation of key tyrosine and threonine residues of p34cdc2 is necessary to turn on its kinase activity. The activity of cdc2 kinase is thus regulated by a combination of translational and post-translational mechanisms. The loss of cdc2 kinase activity at the end of mitosis depends on the destruction of the cyclin subunits. It has been suggested that this destruction is induced by cdc2 kinase itself, thereby providing a negative feedback loop to terminate mitosis. Here we report direct experimental evidence for this idea by showing that cyclin proteolysis can be triggered by adding cdc2 kinase to a cell-free extract of interphase Xenopus eggs.     

Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein.

Oocytes arrested in the G2 phase of the cell cycle contain a p34cdc2/cyclin B complex which is kept in an inactive form by phosphorylation of its p34cdc2 subunit on tyrosine, threonine and perhaps serine residues. The phosphatase(s) involved in p34cdc2 dephosphorylation is unknown, but the product of the fission yeast cdc25+ gene, and its homologues in budding yeast and Drosophila are probably positive regulators of the transition from G2 to M phase. We have purified the inactive p34cdc2/cyclin B complex from G2-arrested starfish oocytes. Addition of the purified bacterially expressed product of the human homologue of the fission yeast cdc25+ gene (p54CDC25H) triggers p34cdc2 dephosphorylation and activates H1 histone kinase activity in this preparation. We propose that the cdc25+ gene product directly activates the p34cdc2-cyclin B complex.     

Fission yeast p107wee1 mitotic inhibitor is a tyrosine/serine kinase.

The fission yeast wee1+ gene product is a dose-dependent, negative regulator of entry into mitosis. wee1+ encodes a protein of relative molecular mass 107,000 (Mr 107K), the C-terminal third of which has strong similarities with the serine/threonine protein kinase family. Here we report that p107wee1 immune complexes phosphorylate p107wee1 equally on serine and tyrosine residues, and also phosphorylate an exogenous substrate, angiotensin II, on tyrosine. Both kinase activities are attributable to p107wee1 because they are also observed when wee1+ is expressed in heterologous systems; both are abolished by a point mutation in the ATP-binding domain, and both behave like an asymmetric monomer of Mr114K on gel filtration and density-gradient centrifugation. Thus the wee1+ gene product is representative of a novel class of protein kinase that phosphorylates both serine and tyrosine residues.     

Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane

The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain.     

Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae.

Progression from G2 to M phase in eukaryotes requires activation of a protein kinase composed of p34cdc2/CDC28 associated with G1-specific cyclins. In some organisms the activation of the kinase at the G2/M boundary is due to dephosphorylation of a highly conserved tyrosine residue at position 15 (Y15) of the cdc2 protein. Here we report that in the budding yeast Saccharomyces cerevisiae, p34CDC28 also undergoes cell-cycle regulated dephosphorylation on an equivalent tyrosine residue (Y19). However, in contrast to previous observations in S. pombe, Xenopus and mammalian cells, dephosphorylation of Y19 is not required for the activation of the CDC28/cyclin kinase. Furthermore, mutation of this tyrosine residue does not affect dependence of mitosis on DNA synthesis nor does it abolish G2 arrest induced by DNA damage. Our data imply that regulated phosphorylation of this tyrosine residue is not the 'universal' means by which the onset of mitosis is determined. We propose that there are other unidentified controls that regulate entry into mitosis.     

Raf-1 activates MAP kinase-kinase.

The normal cellular homologue of the acutely transforming oncogene v-raf is c-raf-1, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases Erk1 and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K). Here we report that MAPK-K as well as Erk1 and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with M(r) 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-1 protooncogene product to be identified.     

Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis

One of the profound changes in cellular morphology which occurs during mitosis is a massive alteration in the organization of the microfilament cytoskeleton. This change, together with other mitotic events including nuclear membrane breakdown, chromosome condensation and formation of mitotic spindles, is induced by a molecular complex called maturation promoting factor. This consists of at least two subunits, a polypeptide of relative molecular mass 45,000-62,000 (Mr 45-62K) known as cyclin, and a 34K catalytic subunit which has serine/threonine kinase activity and is known as cdc2 kinase. Non-muscle caldesmon, an 83K actin- and calmodulin-binding protein, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosphorylation. We now report that cdc2 kinase phosphorylates caldesmon in vitro principally at the same sites as those phosphorylated in vivo during mitosis, and that phosphorylation reduces the binding affinity of caldesmon for both actin and calmodulin. Because caldesmon inhibits actomyosin ATPase, our results suggest that cdc2 kinase directly causes microfilament reorganization during mitosis.     

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.     

Anti-pp60src antibodies are substrates for EGF-stimulated protein kinase

Epidermal growth factor (EGF) stimulates phosphorylation of its own receptor at a tyrosine residue. Similarly, the viral gene product pp60src, which is responsible for cellular transformation by avian sarcoma virus (ASV), phosphorylates itself and immunoglobulin directed against pp60src at tyrosine residues. This unusual site of phosphorylation catalysed by two membrane-associated protein kinases involved in growth control prompted us to study the immunological relatedness of the EGF-stimulated protein kinase and the pp60src. Using anti-pp60src antisera, we attempted to immunoprecipitate the EGF-stimulated protein kinase solubilized from plasma membranes. We report here that neither the EGF-stimulated kinase nor the EGF receptor were immunoprecipitable by anti-pp60src sera. However, anti-pp60src IgG served as a specific substrate for the EGF-stimulated kinase, suggesting a close similarity between the EGF-stimulated kinase and pp60src.     

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.     

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.     

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.     

Dephosphorylation and activation of Xenopus p34cdc2 protein kinase during the cell cycle

Genetic studies in the fission yeast Schizosaccharomyces pombe have established that a critical element required for the G2----M-phase transition in the cell cycle is encoded by the cdc2+ gene. The product of this gene is a serine/threonine protein kinase, designated p34cdc, that is highly conserved functionally from yeast to man2 and has a relative molecular mass of 34,000 (34 K). Purified maturation-promoting factor (MPF) is a complex of p34cdc2 and a 45K substrate that appears in late G2 phase and is sufficient to drive cells into mitosis. This factor has been identified in all eukaryotic cells, and in vitro histone H1 is the preferred substrate for phosphorylation. The increase in the activity of H1 kinase in M-phase is associated with a large increase in total cell protein phosphorylation which is believed to be a consequence of MPF activation. We show here that the H1 kinase activity of p34cdc2 oscillates during the cell cycle in Xenopus, and maximal activity correlates with the dephosphorylated state of p34cdc2. Direct inactivation of MPF in vitro is accompanied by phosphorylation of p34cdc2 and reduction of its protein kinase activity.     

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.     

MAP2 kinase and 70K S6 kinase lie on distinct signalling pathways.

Activation of protein synthesis is required for quiescent cells to transit the cell cycle, and seems to be mediated in part by phosphorylation of the 40S ribosomal protein, S6. A mitogen-activated S6 kinase of relative molecular mass 70,000 (70K) has been isolated from mouse fibroblasts as well as from avian, rat and rabbit tissues. Comparison of complementary DNA sequences shows that this enzyme is distinct from S6 kinase II (92K) found in Xenopus eggs and fibroblasts. Both kinases are activated by serine/threonine phosphorylation, suggesting that at least one serine/threonine kinase links receptor tyrosine kinases with S6 kinases. A candidate for this link is MAP2 kinase, which is rapidly activated by tyrosine/threonine phosphorylation following mitogenic stimulation. Incubation of MAP2 kinase from insulin-treated 3T3-L1 adipocytes with phosphatase-inactivated S6 kinase II from Xenopus leads to partial reactivation and phosphorylation of the enzyme. These and other findings have led to the suggestion that MAP2 kinase also activates the 70K S6 kinase. Here we refute this idea by showing that the two kinases lie on distinct signalling pathways.     

High tyrosine kinase activity in normal nonproliferating cells

Protein phosphorylation at serine and threonine residues has been implicated in the regulation of many cellular processes. More recently, tyrosine residue phosphorylation has been shown to be associated with stimulation of cell proliferation, including viral transformation and stimulation by epidermal growth factors (EGF), platelet-derived growth factor (PDGF) and other compounds related to cellular growth such as insulin and dimethyl sulphoxide. To compare protein kinases and phosphoproteins of normal and leukaemic human haematopoietic cells in vivo and in vitro, we first have investigated the percentages of phosphoserine, phosphothreonine and phosphotyrosine obtained after hydrolysis of proteins from different blood cell fractions phosphorylated in vitro. We report here that phosphotyrosine formed less than 1% of the soluble fractions from polymorphonuclear cells, mononuclear cells (80% circulating lymphocytes, 20% monocytes), blood platelets and red blood cells (not shown). Surprisingly, high percentages of phosphorylated tyrosine were found only in the particulate fractions from non-proliferating anuclear cells, platelets and red blood cells.     

Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery

Polo-like kinase-1 (PLK1) is an essential mitotic kinase regulating multiple aspects of the cell division process. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 kinase domain, but the kinase responsible for this has not yet been affirmatively identified. Here we show that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. We find that aurora A can directly phosphorylate PLK1 on Thr 210, and that activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A (ref. 7). We show that Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A.