Mitogen‐activated protein kinase (MAP kinase) is a 42 kd serine/threonine protein kinase whose enzymatic activity requires phosphorylation of both tyrosyl and threonyl residues. As a step in elucidating the mechanism(s) for activation of this enzyme, we have determined the sites of regulatory phosphorylation. Following proteolytic digestion of 32P‐labeled pp42/MAP kinase with trypsin, only a single phosphopeptide was detected by two‐dimensional peptide mapping, and this peptide contained both phosphotyrosine and phosphothreonine. The amino acid sequence of the peptide, including the phosphorylation sites, was determined using a combination of Fourier transform mass spectrometry and collision‐activated dissociation tandem mass spectrometry with electrospray ionization. The sequence for the pp42/MAP kinase tryptic phosphopeptide is similar (but not identical) to a sequence present in the ERK1‐ and KSS1‐encoded kinases. The two phosphorylation sites are separated by only a single residue. The regulation of activity by dual phosphorylations at closely spaced threonyl and tyrosyl residues has a functional correlate in p34cdc2, and may be characteristic of a family of protein kinases regulating cell cycle transitions.

ABSTRACTpp42, a low-abundance 42-kDa protein, becomes transiently phosphorylated on tyrosine after stimulation of fibroblasts by a variety of mitogens, including epidermal growth factor, platelet-derived growth factor, phorbol 12-myristate 13-acetate, thrombin, and insulin-like growth factor II. The induction of pp42 phosphorylation on tyrosine by such diverse mitogenic agents suggests an important role for pp42 in the cascade of events necessary for cell transition from Go into the cell cycle. However, as with most proteins identified on the basis of their tyrosine phosphorylation, the function of pp42 in cellular regulation is unknown. In this manuscript we report evidence that suggests that pp42 is a serine/threonine-specific protein kinase. Stimulation of 3T3-L1 cells with insulin has been shown to activate a cytosolic serine/threonine kinase capable of phosphorylating microtubule-associated protein 2 (MAP-2) and ribosomal protein S6 kinase H. This cytosolic serine/threonine protein kinase, which itself is phosphorylated on tyrosine, has been termed "MAP kinase." We now report that pp42 phosphorylation and MAP kinase activation occur in fibroblasts in response to similar mitogens, that the two proteins comigrate on one-and two-dimensional polyacrylamide gels, and that the two proteins copurify chromatographically. The major peptides generated from purified MAP kinase by V8 protease digestion are present as a subset of the peptides in digests of pp42 excised from two-dimensional gels. Thus, the results suggest that MAP kinase is tyrosine-phosphorylated pp42.Many growth-factor receptors and oncogene products are tyrosine protein kinases. To understand the mechanisms of intracellular signaling used by these kinases, it will be important to identify and characterize their cellular substrate proteins. Although numerous tyrosine-phosphorylated proteins have been identified by gel electrophoresis in oncogenically transformed or growth factor-stimulated cells, in most cases the biological significance of these phosphorylations has not been determined. pp42 is among the most widely studied of these tyrosine-phosphorylated proteins; it was identified by gel electrophoresis in cells stimulated by any of a number of diverse mitogens [epidermal growth factor (EGF), plateletderived growth factor, insulin-like growth factor II, thrombin, or phorbol 12-myristate 13-acetate (PMA; also called TPA)](1-7) and also has been found in at least some oncogenically transformed cells (8-10). Because ofthe wide variety ofagents that stimulate this phosphorylation, pp42 is believed to be involved in some unknown step in intracellular signaling that is shared by all of these mitogens.Recently a novel serine/threonine protein kinase was identified in insulin-stimulated 3T3-L1 cells (11). This protein kinase has been shown to phosphorylate microtubuleassociated protein 2 (MAP-2) as well as ribosomal protein S6 kinase II in vitro (12). The kinase was also found to migrate on sodium dodecyl sulfate (SDS)/polyacrylamide gels with a molecular ...

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury-induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.

Dorsal root injury results in substantial and often irreversible loss of sensory functions as a result of the limited regenerative capacity of sensory axons and the inhibitory barriers that prevent both axonal entry into and regeneration in the spinal cord. Here, we describe previously unknown effects of the growth factor artemin after crush injury of the dorsal spinal nerve roots in rats. Artemin not only promoted re-entry of multiple classes of sensory fibers into the spinal cord and re-establishment of synaptic function and simple behavior, but it also, surprisingly, promoted the recovery of complex behavior. These effects occurred after a 2-week schedule of intermittent, systemic administration of artemin and persisted for at least 6 months following treatment, suggesting a substantial translational advantage. Systemic artemin administration produced essentially complete and persistent restoration of nociceptive and sensorimotor functions, and could represent a promising therapy that may effectively promote sensory neuronal regeneration and functional recovery after injury.Traumatic injury to the spinal dorsal roots often results in permanent sensory deficits 1,2 . Injured peripheral axons fail to enter the spinal cord at the dorsal root entry zone (DREZ) because of inhibitory barriers and an apparently limited regenerative capacity 2-6 . Oligodendrocytes, astrocytes, microglia and macrophages of the CNS produce growth inhibitory proteins, including Nogo, myelin-associated glycoprotein, and chondroitin sulfate proteoglycans 3,4,7,8 , that can alter the cytoarchitecture of regenerating peripheral axons and can cause growth cone collapse and cessation of growth 3,7,9 . Strategies aimed at altering the hostile central environment to permit axonal regrowth have shown some success. Increasing the levels of neurotrophic factors (for example, neurotrophin-3, nerve growth factor or glial cell line-derived neurotrophic factor, GDNF) by endogenous or exogenous means results in penetration of the DREZ by peripheral axons regenerating locally into the spinal cord 3,10,11 and limited restoration of nociceptive and sensorimotor functions 11 . To date, however, the extent of restoration of sensory functions by growth factors has been incomplete, and growth factors have not promoted recovery of more complex behaviors (for example, touch-evoked

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine protein kinase that becomes enzymatically activated and phosphorylated on tyrosine and threonine following treatment of quiescent cells with a variety of stimulatory agonists. Phosphorylation on both tyrosine and threonine is necessary to maintain full activity, and these two regulatory phosphorylations occur close to each other, separated by a single glutamate. To study the mechanisms by which MAP kinase becomes phosphorylated and activated, we have cloned a full-length cDNA encoding MAP kinase and have expressed the enzyme in Escherichia coli as a soluble nonfusion protein. We find that the enzyme displays a basal, intramolecular autophosphorylation on tyrosine-185 that is accompanied by activation of the enzyme's kinase activity towards an exogenous substrate. The tyrosine-phosphorylated protein displays a small fraction of the activity seen with the fully activated, doubly phosphorylated enzyme isolated from mammalian cells but is activated 10-to 20-fold relative to the unphosphorylated enzyme. These findings raise the possibility that regulation of MAP kinase activity in response to agonist stimulation could occur in part through the enhancement of autophosphorylation on tyrosine.Mitogen-activated protein kinase (MAP kinase) was originally identified as a serine/threonine protein kinase that became phosphorylated on tyrosine and threonine and enzymatically activated following insulin-stimulation of 3T3-L1 cells (1, 2). This enzyme was subsequently found to be identical or closely related to pp42, a protein that becomes tyrosine-phosphorylated in cells treated with various mitogens (3). pp42/MAP kinase recently has been shown to be encoded by a member ofa gene family (4-8). Accordingly, we now refer to it as p2mapk to distinguish it from other members of the family, while still retaining recognizable features of the earlier nomenclature. In addition to being phosphorylated and activated during the Go -* G, transition, p42maPk and/or members ofthis family become phosphorylated and activated during M phase in Xenopus oocytes and in various differentiated, nonmitogenic cells following treatment with stimulatory agonists (9-11). Therefore, we suspect that this enzyme plays a fundamental role in some process common to a number of regulatory events (for review, see ref. 11).Phosphorylation of $2maPk on both tyrosine and threonine is required for it to display full enzymatic activity (12). The sites of regulatory phosphorylation were identified by mass spectrometry and found to be on a single peptide separated by only one amino acid, glutamic acid, in a sequence (ThrGlu-Tyr-Val-Ala-Thr-Arg) that is absolutely conserved in the related protein kinases ERKi (extracellular signal-related protein kinase 1), KSS1, and FUS3 (13). These phosphorylation sites are located just upstream of the Ala-Pro-Glu motif in a region where activating autophosphorylations occur in many other kinases (5, 6, 14).To investigate the mechanism(s) for p42maPk activatio...

The localization of the protein tyrosine kinase pp60c-src to the plasma membrane and to the membrane of secretory vesicles in neurally derived bovine chromaffin cells has suggested that tyrosine phosphorylations may be associated with the process of secretion. In the present study we have identified two cytosolic proteins of approximately 42 and 45 kD that become phosphorylated on tyrosine in response to secretagogue treatment. Phosphorylation of these proteins reached a maximum (3 min after stimulation) before maximum catecholamine release was observed (5-10 min after stimulation). Both secretion and tyrosine phosphorylation of p42 and p45 required extracellular Ca2+. Tyrosine-phosphorylated proteins of similar Mr have previously been identified in 3T3-L1 adipocytes stimulated with insulin (MAP kinase; Ray, L. B., and T. W. Sturgill. 1987. Proc. Natl. Acad. Sci. USA. 84:1502-1506) and in avian and rodent fibroblasts stimulated with a variety of mitogenic agents (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273; Nakamura, K. D., R. Martinez, and M. J. Weber. 1983. Mol. Cell. Biol. 3:380-390). Comparisons of the secretion-associated 42-kD protein of chromaffin cells with the 42-kD protein of Swiss 3T3 fibroblasts and 3T3-L1 adipocytes provide evidence that these three proteins are highly related. This evidence includes comigration during one-dimensional SDS-PAGE, cochromatography using ion exchange and hydrophobic matrices, similar isoelectric points, identical cyanogen-bromide peptide maps, and cochromatography of MAP kinase activity with the tyrosine-phosphorylated form of pp42. This protein(s), which appears to be activated in a variety of cell types, may serve a common function, perhaps in signal transduction involving a cascade of kinases.

(partial) sequence FLTEVVATRWYRAMP, 10 and 8 residues N-terminal from the conserved APE motif (underlined), a general location for autophosphorylations in other kinases (6).Indeed, studies of recombinant p42maPk/ERK2 and p44maPk/ERK1 have revealed that both enzymes undergo slow endogenous phosphorylations on tyrosine and threonine (7-9). The tyrosine phosphorylation is intramolecular and is accompanied by weak activation of its enzymatic activity (8). These observations have been clarified by mass spectrometry and peptide mapping studies, which identified the site of intramolecular tyrosine phosphorylation as Tyr-185, the regulatory tyrosine site, and excluded Thr-183 as a site of significant phosphorylation in recombinant p42maPk (ref. 8 and unpublished data). Endogenous phosphorylation and activation of MAP kinase also occur upon incubation of immunoprecipitates of p42maPk/p44maPk from mammalian cells together with ATP/Mg (10). However, coprecipitation of activating factor(s) cannot be excluded in this case. Thus, plausible mechanisms for activation include enhancement of autophosphorylation at one or both sites in addition to phosphorylation by a Thr-183 and/or Tyr-185 kinase(s), and combinations of these alternatives.Previous studies have pointed to the existence and importance of an upstream protein activator but did not establish the mechanism of activation (11-13). An activating factor(s) was demonstrated by Ahn et al. (11) in fibroblasts treated with epidermal growth factor (EGF) or phorbol esters, with in vitro activation of partially purified mammalian MAP kinase as an assay, and its size was estimated as 50-60 kDa by gel filtration. MAP kinase activator has also been demonstrated in phorbol ester-treated fibroblasts (11) and UM937 cells (12). MAP kinase activator was partially purified from nerve growth factor (NGF)-treated PC12 cells by G6mez and Cohen (13). The NGF-dependent activator was inactivated by phosphatase 2A, but not CD45, suggesting that the activator itself is regulated by serine/threonine phosphorylation (13). Its abilities to induce both threonine and tyrosine phosphorylation of MAP kinase were inactivated in parallel by treatment with phosphatase 2A, suggesting that both were catalyzed by the same protein (13). Finally, strong evidence for a MAP kinase kinase (MAPKK) has been provided in Xenopus laevis by the demonstration that kinase-defective mutants of Xenopus MAP kinase injected into oocytes are phosphorylated on threonine and tyrosine and that mutants with nonphosphorylatable substitutions at sites corresponding to .To define the nature of the MAP kinase activator and provide details of the mechanism in mammalian cells, we have studied in parallel the ability of Mono 5221The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/ threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.The regulation of mammalian cell growth is accomplished by the integration of extracellular signals, such as polypeptide growth factors, with intracellular phosphorylation cascades extending from the plasma membrane to the cell nucleus. Recent studies have indicated that mitogen-activated protein (MAP) kinases (33, 41), also known as extracellular signalregulated kinases (7), are central to these intracellular signal transduction pathways. Two MAP kinases, p42maPk and p44mapk, have been well characterized in many mammalian cell systems (33,41). Both enzymes are phosphorylated on tyrosine and threonine and are activated by a novel protein kinase termed MAP kinase kinase (MKK), also known as MAP kinase/extracellular signal-regulated kinase (MEK) kinase (9,15,29,34,37). The activity of MKK is specific for the regulatory residues, threonine 183 and tyrosine 185, in p42maPk (32), suggesting that MKK is a key regulator of MAP kinases in the cell.The regulation of the MKK/MAP kinase pathway is evolving as a network of crosstalking protein kinases and phosphatases that exert both positive and negative signals. For example, the cellular proto-oncogene product, c-Raf-1, catalyzes the phosphorylation and activation of MKK (10,11,23). Furthermore, the effects of other upstream protein kinases on MKK activity are also becoming apparent. Several groups have demonstrated a link between the MKK pathway and cyclic AMPdependent protein kinase (3,8,17,39,43 protein kinase activity (9,15,29,34,36). Although this method of inactivation has not been documented in vivo, observations suggest that additional phosphorylation(s) of MKK occurs following its initial activation and leads to increased susceptibility to inactivation by phosphatase 2A in vitro (1). Taken together, these observations demonstrate that both protein kinases and phosphatases act upon MKK directly or indirectly after its initial phosphorylation and activation to rigorously control the activity of the MKK/MAP kinase pathway.Recently, two isoforms of MKK, known as MKK1 and MKK2, were identified, and their amino acid sequences were elucidated (9,31,38,45). MKK1 contains a consensus sequence for phosphorylation by p34cdc2, a key regulator of the cell division cyc...