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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stathmin is a ubiquitous, highly conserved 19-kDa cytoplasmic protein whose expression and phosphorylation are regulated in relation to cell proliferation, differentiation or activation, in many biological systems. In this report, we show that stathmin undergoes major phosphorylation in HeLa cells submitted to heat or chemical stress. Heat-shock-induced stathmin phosphorylation was very rapid, as maximal incorporation of phosphate was observed at 5 min. Phosphorylation of stathmin might, therefore, occur as a very early step in the intracellular response to heat shock. The sites of phosphorylation of stathmin involved during the stress response were identified as mostly Ser25 and, to a lesser extent, Ser38. These sites are both followed by a proline residue, and known to be good substrates in vitro for mitogen-activated protein kinase (MAP-kinase) and p34cdc2 kinase, respectively. In lysates from heat-shocked cells, an increased stathmin-kinase activity, distinct from the histone-H1-kinase activity, was found to phosphorylate stathmin mostly on Ser25, the main site for MAP-kinase in vitro. This stathmin-kinase coeluted quantitatively with the stress-activated MAP-kinase from an FPLC MonoQ column. Furthermore, a stathmin kinase activity was precipitated from lysates of heat-shocked HeLa cells by an anti-(MAP-kinase) serum. Together, these results indicate that the phosphorylation of stathmin by MAP-kinase is likely to be a significant component of the signalling array controlling the cellular response to stress, and they further underline the general involvement of stathmin in intracellular signalling.
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PMID:Stathmin is a major substrate for mitogen-activated protein kinase during heat shock and chemical stress in HeLa cells. 785 13

Stathmin, a ubiquitous cytosolic phosphoprotein which may play a role in integrating the effects of diverse signals regulating proliferation, differentiation and other cell functions, was found to be phosphorylated rapidly and stoichiometrically by mitogen-activated protein (MAP) kinase in vitro. Ser-25 was identified as the major site and Ser-38 as a minor site of phosphorylation, while the p42 and p44 isoforms of MAP kinase were the only significant stathmin kinases detected in PC12 cells after stimulation by nerve growth factor (NGF). The results suggest that MAP kinases are the enzymes responsible for increasing the level of phosphorylation of Ser-25, which has been observed previously in PC12 cells following stimulation by NGF.
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PMID:The phosphorylation of stathmin by MAP kinase. 793 47

Stathmin, a probable relay protein possibly integrating multiple intracellular regulatory signals [reviewed in Sobel (1991) Trends Biochem. Sci. 16, 301-305], was expressed in Escherichia coli at levels as high as 20% of total bacterial protein. Characterization of the purified recombinant protein revealed that it had biochemical properties very similar to those of the native protein. It is a good substrate for both cyclic AMP-dependent protein kinase (PKA) and p34cdc2, on the same four sites as the native eukaryotic protein. As shown by m.s., the difference in isoelectric points from the native protein is probably due to the absence of acetylation of the protein produced in bacteria. C.d. studies indicate that stathmin probably contains about 45% of its sequence in an alpha-helical conformation, as also predicted for the sequence between residues 47 and 124 by computer analysis. Replacement of Ser-63 by alanine by in vitro mutagenesis resulted in a ten times less efficient phosphorylation of stathmin by PKA which occurred solely on Ser-16, confirming that Ser-63 is the major target of this kinase. Replacement of Ser-25, the major site phosphorylated by mitogen-activated protein kinase in vitro and in vivo, by the charged amino acid glutamic acid reproduced, in conjunction with the phosphorylation of Ser-16 by PKA, the mobility shift on SDS/polyacrylamide gels induced by the phosphorylation of Ser-25. This result strongly suggests that glutamic acid in position 25 is able to mimic the putative interactions of phosphoserine-25 with phosphoserine-16, as well as the resulting conformational changes that are probably also related to the functional regulation of stathmin.
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PMID:Molecular characterization of human stathmin expressed in Escherichia coli: site-directed mutagenesis of two phosphorylatable serines (Ser-25 and Ser-63). 800 36

Oncoprotein 18 (Op18) has been independently identified due to its increased phosphorylation in response to external signals and its up-regulated expression in acute leukemia. We have identified two serine residues of Op18 that are phosphorylated after triggering by the T cell antigen receptor. One of these residues, Ser25, was shown to be a likely substrate for the mitogen-activated protein (MAP) kinase, while the other residue, Ser16, was shown to be phosphorylated in response to increased intracellular calcium. Our previous site-mapping studies of Op18 also revealed that basal phosphorylation of Op18 is mainly located on Ser38, which was found to be the primary in vitro phosphorylation site of p13suc1-precipitated cdc2 kinase activities. These findings raised the possibility that Op18 may be a substrate for both receptor-regulated calcium-induced protein kinases and the MAP kinase family, as well as being a substrate for the cell-cycle-regulated cdc2 kinase family. In the present report we have performed site-mapping studies of cell-cycle-regulated fluctuations of Op18 phosphorylation. The results reveal that S-phase progression of a synchronised leukemic T cell line is associated with increased phosphorylation of both the Ser25 and Ser38 residues. Moreover, during mitosis, a burst of phosphorylation was observed and at this stage of the cell cycle a major fraction of Op18 was phosphorylated at multiple sites. Phosphorylation of Op18 during mitosis was located primarily on Ser38 and to lesser extent on Ser25, Ser16 and at an unidentified C-terminal residue. In vitro phosphorylation experiments, employing two distinct members of the cdc2 kinase family, were consistent with involvement of both p34-cdc2 and p33-cdk2 in cell-cycle-regulated phosphorylation of Ser25 and Ser38 of Op18. Most importantly, the ratio of Ser25/Ser38 phosphorylation observed in vitro, using either p34-cdc2 or p33-cdk2, was found to be the same as the ratio observed in intact cells during all phases of the cell cycle. These findings suggest that Op18 may be a physiological substrate for several members of the cdc2 kinase family during both the S-phase and the mitotic phase of the cell cycle.
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PMID:Cell-cycle-regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and Ser38. 812 92

A multitude of external signals induce extensive phosphorylation of Oncoprotein 18 (Op18), which suggests a putative role for this protein in signal transduction. We have recently identified two distinct proline-directed kinase families that phosphorylates Op18 with overlapping but distinct site preference. These two kinase families, mitogen-activated protein (MAP) kinases and cyclin-dependent cdc2 kinases, are involved in receptor- and cell cycle-regulated phosphorylation events, respectively. In the present study, site-specific phosphorylation of Op18 in response to stimulation of the antigen receptor-associated CD3 complex was analyzed in the Jurkat T cell-line. The results show that CD3-induced phosphorylation of Ser-25 of Op18, which is the primary MAP kinase phosphorylation site, can be induced by an apparently protein kinase C (PKC)-independent signal transduction pathway. We also demonstrate that Ser-16 of Op18 is specifically phosphorylated in response to the Ca2+ signal generated by CD3 stimulation or by the Ca2+ ionophore ionomycin. Ser-16 phosphorylation occurs independently of both PKC and MAP kinase activation. Using site-specific Op18 mutants and tryptic phosphopeptide mapping, we show that phosphorylation of Ser-16 of Op18 together with Ser-25, or Ser-25 and Ser-38, generates two Op18 phosphoisomers showing a dramatic electrophoretic retardation. In conclusion, site-mapping studies of Op18 reveal that CD3 stimulation results in an apparently PKC-independent activation of both the MAP kinase and a Ca(2+)-regulated kinase pathway, which results in phosphorylation of distinct sites of Op18. The data also pinpoints the specific phosphorylation events that result in electrophoretic retardation of Op18.
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PMID:Multiple signal transduction pathways induce phosphorylation of serines 16, 25, and 38 of oncoprotein 18 in T lymphocytes. 824 3

Oncoprotein 18 (Op18) is an 18-19-kDa cytoplasmic phosphoprotein, of unknown function, that is frequently up-regulated in transformed cells. Stimulation of various cell-surface receptors results in extensive phosphorylation of Op18 and this protein has, therefore, previously been implicated in intracellular signaling. In the present study, by expression of specific Op18 cDNA mutant constructs and phosphopeptide mapping, we have identified in vivo phosphorylation sites. In conjunction with in vitro phosphorylation experiments, using purified wild-type and mutant Op18 proteins in combination with a series of kinases, these results have identified two distinct proline-directed kinase families that phosphorylate Op18 with overlapping but distinct site preference. These two kinase families, mitogen activated protein (MAP) kinases and cyclin dependent cdc2 kinases, are involved in receptor and cell cycle-regulated phosphorylation events, respectively. Therefore, Op18 may reside at a junction where receptor and cell cycle-regulated kinase families interact with the same substrate. The present study shows that the MAP kinase has a 20-fold preference for Ser25 as opposed to Ser38 of Op18, while cdc2 kinases have a 5-fold preference for the Ser38 residue. Only a minor fraction of the 4.5 x 10(6) Op18 molecules/cell in a leukemic T-cell line are normally in their Ser25 phosphorylated form. However, antigen receptor stimulation of this cell line is shown to result in a rapid conversion of 35-45% of all Op18 molecules to the Ser25 phosphorylated form. These results suggest that Ser25 of Op18 may be a major cytoplasmic target for the MAP kinase in cells with high expression of Op18.
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PMID:Serine 25 of oncoprotein 18 is a major cytosolic target for the mitogen-activated protein kinase. 832 80

Stathmin is a ubiquitous, highly conserved phosphoprotein which most likely acts as a relay integrating various intracellular pathways regulating cell proliferation, differentiation, and functions. At least 14 molecular forms of stathmin have been identified so far, which migrate as 2 unphosphorylated and 12 increasingly phosphorylated spots (M(r) = 19,000-23,000; pI = 6.2-5.6) on two-dimensional electrophoretic gels, and whose pattern may reflect the state of activation of cells. We found that stathmin could be phosphorylated in vitro by at least three different protein kinases: cAMP-dependent protein kinase, p34cdc2, and casein kinase II, cAMP-dependent protein kinase catalyzed the phosphorylation of stathmin on serines 16 (K-R-A-S) and 63 (R-R-K-S), whereas p34cdc2 induced phosphorylation on serines 25 (I-L-S-P-R) and 38 (P-L-S-P-P-K-K-K). Interestingly, phosphorylation by both kinases together yielded all of the phosphoforms of stathmin identified so far. Two-dimensional phosphopeptide analysis allowed us to demonstrate that the same four sites were exclusively found to be phosphorylated in vivo, in brain tissue as well as in control or nerve growth factor-stimulated PC12 cells. In this latter case, the major site phosphorylated in response to nerve growth factor being serine 25, it is likely that a kinase such as a mitogen-activated protein kinase, known to be activated by growth factors, might directly phosphorylate stathmin. The phosphopeptide map analysis allowed further identification of the specific combinations among the four sites whose phosphorylation is responsible for the characteristic two-dimensional polyacrylamide gel electrophoresis migration of the resulting stathmin forms both in vitro and in vivo and revealed the existence of likely structural interactions between the sites phosphorylated. In conclusion, our results show that phosphorylation of serines 16, 25, 38, and 63 accounts for all of the major functional stathmin forms observed in vivo. The present identification of these sites will foster a better understanding of some intracellular mechanisms involved in the diverse physiological regulation of the proliferation, differentiation, and functions of cells, including the role of stathmin in these processes as a relay integrating diverse signaling pathways.
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PMID:Multiple phosphorylation of stathmin. Identification of four sites phosphorylated in intact cells and in vitro by cyclic AMP-dependent protein kinase and p34cdc2. 837 65

Stathmin is a ubiquitous cytosolic protein which undergoes extensive phosphorylation in response to a variety of external signals. It is highly abundant in developing neurons. The use of antisense oligonucleotides which selectively block stathmin expression has allowed us to study directly its role in rat PC12 cells. We show that stathmin depletion prevents nerve growth factor (NGF)-stimulated differentiation of PC12 cells into sympathetic-like neurons although the expression of several NGF-inducible genes was not affected. Furthermore, we found that stathmin phosphorylation in PC12 cells which is induced by NGF depends on mitogen-activated protein kinase (MAPK) activity. We conclude that stathmin is an essential component of the NGF-induced MAPK signaling pathway and performs a key role during differentiation of developing neurons.
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PMID:The phosphoprotein stathmin is essential for nerve growth factor-stimulated differentiation. 868 72

The effects of a pan-CD45 mAb (CD45.2) on TCR-mediated signaling pathways were investigated in Jurkat T cells. The simultaneous addition of CD45 mAb with an activating OKT3 mAb had little effect on TCR-stimulated signals. However, when Jurkat cells were exposed to the CD45 mAb for 10 to 20 min before the addition of OKT3, a partial uncoupling of the TCR from intracellular signals was observed. The maximal increase in intracellular calcium was inhibited 47 +/- 10% (n = 11, range 33-67%), whereas no inhibition of inositol trisphosphate production was detected. The transient TCR-mediated activation of the Ca2+/calmodulin-activated kinase IV/Gr was also inhibited by the CD45 mAb, and this was reflected in a 50 to 60% inhibition in the TCR-stimulated generation of the p21 and p23 phosphoisomers of oncoprotein 18, a Ca2+/calmodulin-activated kinase IV/Gr substrate recently implicated in cell cycle regulatory events. Oncoprotein 18 is also a substrate for mitogen- activated protein kinase, but no inhibition by the CD45 mAb of TCR-triggered mitogen-activated protein kinase activation was observed. The CD45 mAb was therefore selective in causing the uncoupling of the TCR from calcium signals and calcium-regulated events without promoting a general inhibition of all TCR-mediated signals. Confocal microscopy revealed that binding of the CD45 mAb caused patching of CD45 molecules at the cell surface and, unexpectedly, a marked redistribution of intracellular CD45. However, no change was observed in the total level of CD45 expressed at the cell surface. Aggregation of CD45 at the cell surface may result in its sequestration from its tyrosine kinase substrates, with a consequent selective uncoupling of the TCR from intracellular signaling pathways.
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PMID:CD45 monoclonal antibodies inhibit TCR-mediated calcium signals, calmodulin-kinase IV/Gr activation, and oncoprotein 18 phosphorylation. 868 2

Prolactin induces milk protein gene expression in rabbit primary mammary cells without any concomitant cell multiplication. Prolactin or other lactogenic hormones is the major inducer of cell division in the rat lymphoid Nb2 cells. In Nb2 cells, prolactin also rapidly induces the expression of the c-myc gene, and beta-actin and stathmin gene expression is induced more slowly. The possible involvement of casein kinase II (CKII), mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in these process is not well known. The present work was undertaken to evaluate the effect of prolactin on these protein kinases and to determine the possible involvement of these enzymes in the activity of several genes under the control of the hormone. In rabbit mammary cells, prolactin did not alter CKII activity but did transiently stimulate MAP kinase activity. Prolactin also stimulated Ca(2+)-independent PKC. This effect was visible after 10 min and was maintained for at least 24 hr. Staurosporine, an inhibitor of PKC and of several tyrosine kinases altered Ca(2+)-independent PKC only moderately. In contrast, GF 109203X, a potent and specific inhibitor of PKC, abrogated almost all PKC activity. Staurosporine, but not GF 109203X, prevented the induction of the casein gene by prolactin. In Nb2 cells, prolactin induced a slow stimulation of CKII activity. The hormone did not induce MAP kinase activity. Prolactin stimulated Ca(2+)-independent PKC over periods of 24 hr. GF 109203X, but not staurosporine, inhibited PKC activity, whereas staurosporine but not GF 109203X, inhibited the induction of Nb2 cell multiplication and the accumulation of c-myc, beta-actin and stathmin mRNAs. From these data, it can be concluded that (1) the stimulation of CKII by prolactin in Nb2 cells is concomitant with cell multiplication: (2) MAPK stimulation is not necessary for prolactin to induce Nb2 cell multiplication; and (3) PKC is stimulated in mammary and Nb2 cells, but this stimulation is not required for prolactin to stimulate casein, c-myc, beta-actin and stathmin gene expression and Nb2 cell division.
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PMID:The effect of prolactin on casein kinase II, MAP kinase and PKC in rabbit mammary cells and Nb2 rat lymphoid cells. 898 34


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