Defective regulation of mitogen-activated protein kinase activity in a 3T3 cell variant mitogenically nonresponsive to tetradecanoyl phorbol acetate.

Mitogen-activated protein (MAP) kinase is a serine/threonine-specific protein kinase which is activated in response to various mitogenic agonists (e.g., epidermal growth factor, insulin, and the tumor promoter tetradecanoyl phorbol acetate [TPA]) and requires both threonine and tyrosine phosphorylation for activity. This enzyme has recently been shown to be identical or closely related to pp42, a protein which becomes tyrosine phosphorylated in response to mitogenic stimulation. Neither the kinases which regulate MAP kinase/pp42 nor the in vivo substrates for this enzyme are known. Because MAP MAP kinase is activated and phosphorylated in response both to agents which stimulate tyrosine kinase receptors and to agents which stimulate protein kinase C, a serine/threonine kinase, we have examined the regulation and phosphorylation of this enzyme in 3T3-TNR9 cells, a variant cell line partially defective in protein kinase C-mediated signalling. In this communication, we show that in the 3T3-TNR9 variant cell line, TPA does not cause the characteristically rapid phosphorylation of pp42 or the activation and phosphorylation of MAP kinase. This defective response is not due to the absence of the MAP kinase/pp42 protein itself because both tyrosine phosphorylation of MAP kinase/pp42 and its enzymatic activation could be induced by platelet-derived growth factor in the 3T3-TNR9 cells. Thus, the defect in these variant cells apparently resides in some aspect of the regulation of MAP kinase phosphorylation. Since the 3T3-TNR9 cells are also defective with respect to the TPA-induced increase in ribosomal protein S6 kinase, these in vivo results reinforce the earlier in vitro finding that MAP kinase can regulate S6 kinase activity. These findings suggest a key role for MAP kinase in a kinase cascade cascade involved in the control of cell proliferation.     

Cell cycle reentry of mammalian fibroblasts is accompanied by the sustained activation of P44mapk and P42mapk isoforms in the G1 phase and their inactivation at the G1/s transition

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that are rapidly activated in response to mitogenic stimuli. Here we examined the enzymatic activity and phosphorylation state of the individual p44^mapk and p42^mapk isoforms during early G1 and late G1 phase of the mammalian cell cycle. Release of fibroblast cells from early G1 block was accompanied by a rapid rise in the myelin basic protein (MBP) kinase activity of p44^mapk and p42^mapk, which declined slowly over several hours to reach negligible values as cells enter S phase. When cells were released from late G1 block, the activity of p44^mapk and p42^mapk increased transiently, and then rapidly declined to baseline values during G1 to S phase transition. Cells released at the G1/S boundary in a medium lacking growth factors entered S phase in the complete absence of MAP kinase activity. Unlike MAP kinases, the histone H1 kinase activity of p33^cdk2 was elevated in late G1-arrested cells and continued to increase during S phase entry. The enzymatic activation of p44^mapk and p42^mapk in both early G1 and late G1 phase was accompanied by an increase in the phosphothreonine and phosphotyrosine content of the proteins. These findings suggest that the sustained activation of MAP kinases during G1 progression and their inactivation at the G1/S transition are two regulatory processes involved in the mitogenic response to growth factors. © 1995 Wiley-Liss, Inc.     

Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts

Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.     

A mitogen-activated protein (MAP) kinase activating factor in mammalian mitogen-stimulated cells is homologous to Xenopus M phase MAP kinase activator.

The mitogen-activated protein (MAP) kinases, a family of 40-45-kDa kinases whose activation requires both tyrosine and threonine/serine phosphorylations, are suggested to play key roles in various phosphorylation cascades. A previous study of Krebs and co-workers (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) detected an activity in epidermal growth factor (EGF)-stimulated 3T3 cells that can stimulate inactive MAP kinases. We observed this activity in rat 3Y1 cells treated with various mitogenic factors and in PC12 cells treated with nerve growth factor (NGF). Its kinetics of activation and deactivation following EGF or NGF stimulation roughly paralleled that of MAP kinase. The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+ and Mg2+ and was inactivated by phosphatase 2A treatment in vitro. This activator has been isolated from EGF-stimulated 3Y1 cells by sequential chromatography and identified as a 45-kDa monomeric protein. It was able to activate mammalian and Xenopus MAP kinases in vitro and was very similar to Xenopus M phase MAP kinase activating factor, which was purified previously from mature oocytes (Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982), in terms of its functional, immunological, and physicochemical properties. Thus, the same or a similar upstream activating factor may function in mitogen-induced and M phase-promoting factor-induced MAP kinase activation pathways.     

Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase.

Epidermal growth factor stimulates the activity of several cytosolic serine/threonine protein kinases in quiescent Swiss 3T3 cells. Two of these, which use myelin basic protein (MBP) as substrate, act as kinase kinases in that they are able to activate a separate peptide kinase activity in vitro by a mechanism involving protein phosphorylation. In this study, we have identified two activities from extracts of epidermal growth factor-treated cells that stimulate an ATP-dependent activation of both of the MBP kinases, derived in their inactive precursor forms from extracts of untreated cells. The resulting MBP kinase activities are stable to further purification and can be inactivated with either tyrosine or serine/threonine protein phosphatases and then reactivated to their original levels of activity. Thus, we propose that the in vitro activation involves protein phosphorylation, stimulated by the action of novel MBP kinase activating factors that represent intermediate components in a growth factor-stimulated kinase cascade.     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine phosphorylation is an obligatory event in IL-2 secretion.

Activation of T lymphocytes leads to the production of the T cell growth factor IL-2 that regulates T cell proliferation. This activation is associated with several potential intracellular signalling events including increased activity of phospholipase C (PLC) and resultant increases in production of inositol phosphates and diacylglycerols. In addition, phosphorylation of specific intracellular proteins on serine, threonine, and tyrosine residues increases. The role of each of these events in IL-2 production is unclear. Using Western blotting with antiphosphotyrosine antibodies, we demonstrate that activation of murine T cells with mitogenic lectins or anti-CD3 antibodies leads to a rapid increase in tyrosine phosphorylation of proteins of 120, 72, 62, 55, and 40 kDa. Similar patterns of antiphosphotyrosine antibodies reactivity were observed in splenocytes, a T cell hybridoma, and a T lymphoma. Tyrosine phosphorylation was detectable within minutes of addition of mitogenic lectins and persisted for at least 6 h. Pretreatment of the cells with pertussis toxin did not inhibit tyrosine phosphorylation indicating that a pertussis toxin-sensitive G protein is not involved in signal transduction. Neither increasing cytosolic-free calcium nor activating protein kinase C mimicked the effects of mitogenic lectins suggesting that tyrosine phosphorylation was not a consequence of activation of PLC. This was confirmed by demonstrating that mitogenic lectins induced similar patterns of tyrosine phosphorylation in cells in which activation of the TCR leads to increased PLC activity and in cells in which PLC is not stimulated. To test whether tyrosine phosphorylation is linked to IL-2 secretion, we determined the effect of three specific tyrosine kinase inhibitors (tyrphostins) on tyrosine phosphorylation, IL-2 secretion, and cellular proliferation. The concentration dependence of inhibition of tyrosine phosphorylation and IL-2 production were similar. However, higher concentrations of the tyrphostins were required to inhibit constitutive proliferation of the T cell line indicating that inhibition of IL-2 secretion was not secondary to nonspecific toxic effects of the tyrphostins. Addition of the tyrphostins after mitogenic lectin decreased the amount of tyrosine phosphorylation and IL-2 secretion in parallel. This indicates that both tyrosine kinases and phosphatases are activated and that continuous tyrosine phosphorylation is likely required for IL-2 secretion. Therefore, tyrosine phosphorylation appears to represent an obligatory event in the transmembrane signaling processes that lead to IL-2 secretion.     

Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. The role of lyso-PAF disposal and free arachidonic acid.

Tumour necrosis factor (TNF) is a potent mitogen for some fibroblast cell lines. Here we have examined the TNF-mediated changes in protein phosphorylation in Swiss 3T3 and human FS-4 fibroblasts, and compared them with changes observed after the treatment of cells with other mitogens, such as platelet-derived growth factor (PDGF) and bombesin. TNF stimulated the rapid phosphorylation of two 41,000-Mr and two 43,000-Mr cytosol proteins on tyrosine, threonine and/or serine, as did PDGF, epidermal growth factor and fibroblast growth factor; the increased levels of this mitogen-induced protein-tyrosine phosphorylation correlated well with the extent of mitogen-induced DNA synthesis as determined by the percentage of labelled nuclei. In contrast, bombesin, which is an even better mitogen for Swiss 3T3 cells than TNF, stimulated the tyrosine phosphorylation of 41,000-Mr and 43,000-Mr proteins only to a limited extent. On the other hand, bombesin and PDGF stimulated the rapid serine phosphorylation of an 80,000-Mr acidic protein, a major substrate for protein kinase C; increased phosphorylation of the 80,000-Mr protein was not observed at all when cells were stimulated with TNF. These results suggest significant differences among the mitogenic signalling pathways of TNF, PDGF and bombesin as regards the involvement of protein kinases; the mitogenic signalling pathway of TNF involves the activation of tyrosine kinase, but not of protein kinase C, whereas bombesin seems to transduce its mitogenic signal mainly through the activation of protein kinase C, and the activation of both kinases seems to be involved in the mitogenic signalling pathway of PDGF.     

Both ERK1 and ERK2 kinases promote G2/M arrest in etoposide-treated MCF7 cells by facilitating ATM activation

The MEK–ERK pathway plays a role in DNA damage response (DDR). This has been thoroughly studied by modulating MEK activation. However, much less has been done to directly examine the contributions of ERK1 and ERK2 kinases to DDR. Etoposide induces G2/M arrest in a variety of cell lines, including MCF7 cells. DNA damage-induced G2/M arrest depends on the activation of the protein kinase ataxia-telangiectasia mutated (ATM). ATM subsequently activates CHK2 by phosphorylating CHK2 threonine 68 (T68) and CHK2 inactivates CDC25C via phosphorylation of its serine 216 (S216), resulting in G2/M arrest. To determine the contribution of ERK1 and ERK2 to etoposide-induced G2/M arrest, we individually knocked-down ERK1 and ERK2 in MCF7 cells using specific small interfering RNA (siRNA). Knockdown of either kinases significantly reduced ATM activation in response to etoposide treatment, and thereby attenuated phosphorylation of the ATM substrates, including the S139 of H2AX (γH2AX), p53 S15, and CHK2 T68. Consistent with these observations, knockdown of either ERK1 or ERK2 reduced etoposide-induced CDC25C S216 phosphorylation and significantly compromised etoposide-induced G2/M arrest in MCF7 cells. Taken together, we demonstrated that both ERK1 and ERK2 kinases play a role in etoposide-induced G2/M arrest by facilitating activation of the ATM pathway. These observations suggest that a cellular threshold level of ERK kinase activity is required for the proper checkpoint activation in MCF7 cells.     

Analysis of the protein kinase activity of moss phytochrome expressed in fibroblast cell culture

In the moss Ceratodon purpureus a phytochrome gene encodes a phytochrome type (PhyCer) which has a C-terminal domain homologous to the catalytic domain of eukaryotic protein kinases (PKs). PhyCer exhibits sequence conservation to serine/ threonine as well to tyrosine kinases. Since PhyCer is expressed very weakly in moss cells, to investigate the proposed PK activity of PhyCer, we overexpressed PhyCer transiently in fibroblast cells. For this purpose we made a chimeric receptor, EC-R, which consists of the extracellular, the membrane-spanning and the juxtamembrane domains of the human epidermal growth-factor receptor (EGF-R) linked to the PK catalytic domain of PhyCer (CerKin). The expression of EC-R in transiently transfected cells was confirmed with antibodies directed against the extracellular domain of EGF-R or against CerKin. Both EGF-R and EC-R were immunoprecipitated from lysates of overexpressing cells with antibodies against the extracellular domain of EGF-R. Phosphorylation experiments were performed with the immunoprecipitates and the phosphorylation products were subjected to phosphoamino acid analysis. Phosphorylation products specifically obtained with EC-R-transfected cells exhibit phosphorylation on serine and threonine residues. In EC-R transfected cells the endogenous EGF-R showed enhanced phosphorylation of serine and threonine residues compared to EGF-R immuno-precipitated from control cells. Although CerKin is closest to the catalytic domain of a protein tyrosine kinase from Dictyostelium discoideum, EC-R does not appear to phosphorylate tyrosine residues in vitro. From our data we conclude that PhyCer carries an active PK domain capable of phosphorylating serine and threonine residues.Abbreviations CerKin protein kinase catalytic domain of PhyCer - EC-R chimeric receptor consisting of the extracellular, the membrane spanning and the juxtamembrane domains of the human epidermal growth factor receptor (EGF-R) linked to the protein kinase catalytic domain of PhyCer - EGF-R epidermal growth factor receptor - mAb monoclonal antibody - PhyCer phytochrome gene in Ceratodon encoding a phytochrome type which has a C-terminal domain homologous to the catalytic domain of eucaryotic protein kinases - PK protein kinase - PVDF polyvinyl difluoride - Ser serine - Thr threonine - Tyr tyrosine Dr. Patricia Algarra was supported by the Alexander von Humboldt Foundation, Germany. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany.     

Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor.

FGF-2 exerts its pleiotropic effects on cell growth and differentiation by interacting with specific cell surface receptors. In addition, exogenously added FGF-2 is translocated from outside the cell to the nucleus during G1-S transition. In this study, we show that a single point mutation in FGF-2 (substitution of residue serine 117 by alanine) is sufficient to drastically reduce its mitogenic activity without affecting its differentiation properties. The FGF-2(S117A) mutant binds to and activates tyrosine kinase receptors and induces MAPK and p70S6K activation as strongly as the wild-type FGF-2. We demonstrate that this mutant enters NIH3T3 cells, is translocated to the nucleus, and is phosphorylated similar to the wild-type growth factor. This suggests that FGF-2 mitogenic activity may require, in addition to signaling through cell surface receptors and nuclear translocation, activation of nuclear targets. We have previously shown that, in vitro, FGF-2 directly stimulates the activity of the casein kinase 2 (CK2), a ubiquitous serine/threonine kinase involved in the control of cell proliferation. We report that, in vivo, FGF-2(WT) transiently interacts with CK2 and stimulates its activity in the nucleus during G1-S transition in NIH3T3 cells. In contrast, the FGF-2(S117A) mutant fails to interact with CK2. Thus, our results show that FGF-2 mitogenic and differentiation activities can be dissociated by a single point mutation and that CK2 may be a new nuclear effector involved in FGF-2 mitogenic activity.-Bailly, K., Soulet, F., Leroy, D., Amalric, F., Bouche, G. Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor (FGF-2).     

Phosphorylation of elongation factor G and ribosomal protein S6 in bacteriophage T7-infected Escherichia coli

Bacteriophage T7 expresses a serine/threonine-specific protein kinase activity during Infection of Its host, Escherichia coli. The protein kinase (gpO.7 PK), encoded by the T7 early gene 0.7, enhances phage reproduction under sub-optimal growth conditions. It was previously shown that ribosomal protein S1 and translation initiation factors IF1, IF2, and IF3 are phosphoryiated in T7-infected cells, and it was suggested that phosphorylation of these proteins may serve to stimulate translation of the phage late mRNAs. Using high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation, we show that elongation factor G and ribosomal protein S6 are phosphorylated following T7 infection. The gel electro-phoretic data moreover indicate that elongation factor P is phosphorylated in T7-infected cells. T7 early and late mRNAs are processed by ribonuclease III, whose activity is stimulated through phosphorylation by gp0.7 PK. Specific overexpression and phosphorylation was used to locate the RNase III polypeptide in the standard two-dimensional gel pattern, and to confirm that serine is the phosphate-accepting amino acid. The two-dimensional gels show that the in vivo expression of gp0.7 PK results in the phosphorylation of over 90 proteins, which Is a significantly higher number than previous estimates. The protein kinase activities of the T7-related phages T3 and BA14 produce essentially the same pattern of phosphorylated proteins as that of T7. Finally, several experimental variables are analysed which influence the production and pattern of phosphorylated proteins in both uninfected and T7-rnfected cells.     

Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells

Growth factor activation of serine/threonine protein kinases was studied by treating quiescent Swiss 3T3 cells with epidermal growth factor (EGF) and examining cytosolic extracts for protein kinase activity under conditions inhibitory to calcium- and cyclic nucleotide-dependent kinases. Cytosolic extracts of cells stimulated for 5 min were fractionated by Mono Q fast protein liquid chromatography. Eight peaks of kinase activity were resolved, of which five were stimulated by EGF treatment of cells. These peaks were revealed using the synthetic peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (S6 peptide), 40 S ribosomal S6 protein, glycogen synthase, microtubule-associated protein 2, and myelin basic protein as substrates. The peaks varied in the kinetics of their activation by EGF and in their response to insulin. Selected peaks were resolved further by sizing gel chromatography. The results together indicate that at least seven distinct fractions of cytosolic kinase activities are stimulated in Swiss 3T3 cells by EGF. One of these, which phosphorylates both S6 protein and S6 peptide, is similar to the S6 kinase characterized previously in this cell line by others. Four additional activities that also phosphorylate the S6 protein and S6 peptide appear unrelated to this enzyme. Finally, two kinase activities that phosphorylate both myelin basic protein and microtubule associated protein 2 are EGF stimulated. One is similar to an insulin-stimulated microtubule-associated protein 2 kinase described in other cell lines whereas the other seems to represent a novel activity. Several of these EGF-stimulated activities were inactivated by protein phosphatases, suggesting that they might be regulated by phosphorylation.     

The role of PIM kinases in human and mouse CD4+ T cell activation and inflammatory bowel disease

PIM kinases are a family of three serine/threonine kinases expressed following T cell activation. Using potent selective small molecule antagonists of PIM-1/3 kinases, we demonstrate a potential role for these enzymes in naïve and effector CD4+ T cell activation. PIM-1/3 inhibition prevented CD4+ T cell proliferation by inducing a G0/G1 cell cycle arrest without affecting cellular survival. In the absence of PIM-1/3 kinase activity, naïve CD4+ T cells failed to fully differentiate into effector cells both in vitro and in vivo. Therapeutic dosing of a PIM-1/3 inhibitor was efficacious in a CD4+ T cell-mediated model of inflammatory bowel disease suggesting that PIM-1 and PIM-3 kinase activity contributes to sustained disease severity. These results demonstrate that PIM-1/3 kinases have an important role in CD4+ T cell responses and inhibition of this activity may provide a therapeutic benefit in T cell-mediated diseases.     

Phosphorylation of Escherichia coli translation initiation factors by the bacteriophage T7 protein kinase.

The lytic coliphage T7 encodes a serine/threonine-specific protein kinase which supports viral reproduction under suboptimal growth conditions. Expression of the protein kinase in T7-infected Escherichia coli cells results in the phosphorylation of 30S ribosomal subunit protein S1, and initiation factors IF1, IF2, and IF3, as determined by high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation analysis. Phosphorylation occurs either exclusively on threonine (IF1, IF3, S1) or on serine and threonine (IF2). There is no phosphorylation of these proteins in uninfected cells or in cells infected with T7 lacking the protein kinase function. Phosphorylation of the initiation factors coincides with the onset of T7 late protein synthesis, occurring 9-12-min postinfection. T7 late protein synthesis, otherwise inhibited in ColIb plasmid-containing cells, is specifically supported by expression of the protein kinase. These results provide the first evidence for the functional involvement of protein phosphorylation in the control of bacterial translation.     

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.     

Okadaic acid stimulates the activity of the nerve growth factor-sensitive S6 kinase of PC12 cells.

PC12 pheochromocytoma cells contain at least two different and separable kinases that phosphorylate the S6 protein of the ribosomes. The activity of one of these S6 kinases is increased by treatment of the cells with nerve growth factor and of the other by treatment with epidermal growth factor. Okadaic acid increases the activity of the nerve growth factor-sensitive S6 kinase. The data suggest that the nerve growth factor-sensitive S6 kinase is activated by phosphorylation on serine or threonine residues and is inactivated by either phosphatase 1 or phosphatase 2A, probably the latter.     

Evidence for an epidermal growth factor-stimulated protein kinase cascade in Swiss 3T3 cells. Activation of serine peptide kinase activity by myelin basic protein kinases in vitro

Two intermediary kinases in a protein serine/threonine kinase cascade that is triggered in the response of Swiss 3T3 cells to epidermal growth factor (EGF) have been identified. Several separable EGF-stimulated serine/threonine kinase activities were characterized in the preceding paper (Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E.G. (1990) J. Biol. Chem. 265, 11487-11494). These were preincubated in various combinations in the presence of MgATP with chromatographic fractions from unstimulated cell extracts. Activation of the rate of phosphorylation of a synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, was observed on preincubation of the breakthrough fraction from unstimulated cell extracts with either of two distinct EGF-stimulated kinase activities, each of which phosphorylated myelin basic protein. Kinetic analysis and fractionation by sizing gel chromatography demonstrated that two myelin basic protein kinase activities (of approximately 30 and approximately 50 kDa) represented the activating components in the mixtures whereas the unstimulated cell extract breakthrough gave rise in each case to the activated Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala peptide kinase activity of approximately 110 kDa. Inasmuch as the in vitro activation reactions required magnesium plus ATP and were reversed by protein phosphatase treatment, an activation mechanism involving phosphoryl transfer is suggested.