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.

Background-Hyperhomocysteinemia is a putative risk factor for cardiovascular disease, which also impairs endothelium-dependent vasodilatation. A number of other risk factors for cardiovascular disease may exert their adverse vascular effects in part by elevating plasma levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase. Accordingly, we determined if homocysteine could increase ADMA levels. Methods and Results-When endothelial or nonvascular cells were exposed to DL-homocysteine or to its precursor L-methionine, ADMA concentration in the cell culture medium increased in a dose-and time-dependent fashion. This effect was associated with the reduced activity of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA. Furthermore, homocysteine-induced accumulation of ADMA was associated with reduced nitric oxide synthesis by endothelial cells and segments of pig aorta. The antioxidant pyrrollidine dithiocarbamate preserved DDAH activity and reduced ADMA accumulation. Moreover, homocysteine dose-dependently reduced the activity of recombinant human DDAH in a cell free system, an effect that was due to a direct interaction between homocysteine and DDAH. Conclusion-Homocysteine

We have defined inactive ␣ and fragments of ␤-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments. Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the ␣ fragment increased complemented enzyme activity by up to 4 orders of magnitude. ␤-Lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate. The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date. P rotein-protein interactions are involved in every cellular process ranging from gene expression and signal transduction to cell division and differentiation, yet they have been among the most difficult aspects of cell biology to study. Standard biochemical methods have yielded most of the available information about such interactions, but these assays often are limited by the available reagents such as monoclonal antibodies for immunoprecipitation or lack the appropriate cellular context.The development of fusion protein-based assays such as the yeast two-hybrid method (1) has expanded the potential for studying protein interactions in intact cells greatly. However, this assay relies on the transcription of a reporter gene; consequently it is not applicable to studies of the kinetics of protein-protein interactions and is unable to detect the interaction of compartmentalized proteins such as receptors at the cell surface. A method based on fluorescence resonance energy transfer provided a further advance and currently is one of the most accurate methods used to monitor dynamic interactions (2). However, the incremental changes in fluorescence assayed by fluorescence resonance energy transfer are small, and the stringent steric requirements for detecting the interacting proteins can restrict the utility of this technique.Assays based on the complementation of enzyme fragments fused to interacting proteins that regenerate enzymatic activity after dimerization are particularly well suited for monitoring inducible protein interactions (reviewed in ref.3). These systems have important advantages including low-level expression of the test proteins, generation of signal as a direct result of the interaction, and enzymatic amplification. As a result, they are highly sensitive and physiologically relevant assays (4). Additionally, assays based on enzyme complementation can be performed in any cell type of interest or in diverse cellular compartments such as the nucleus, secretory vesicles, or plasma membrane.The class A ␤-lactamases are particularly attractive candidates for an assay based on enzyme fragment complementation because of the fact that they are monomeric and of relatively small si...

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...

p42mapk [mitogen activated protein (MAP) kinase; extracellular signal-regulated protein kinase (ERK)] is a serine/threonine-specific protein kinase that is activated by dual tyrosine and threonine phosphorylation in response to diverse agonists. Both the tyrosine and threonine phosphorylations are necessary for full enzymic activity. A MAP kinase activator recently purified and cloned has been shown to be a protein kinase (MAP kinase kinase) that is able to induce the dual phosphorylation of MAP kinase on both the regulatory tyrosine and threonine sites in vitro. In the present paper we have utilized MAP kinase mutants altered in the sites of regulatory phosphorylation to show, both in vivo and in vitro, that phosphorylation of the tyrosine and the threonine can occur independently of one another, with no required order of phosphorylation. We also utilized kinase-defective variants of MAP kinase with mutations in either the ATP-binding loop or the catalytic loop, and obtained data suggesting that the activity or structure of the catalytic loop of MAP kinase plays an important role in its own dual phosphorylation.

EMBL accession no. X58712Pp42/MAP kinase (refered to here as p42maPk) becomes

p42/microtubule-associated protein kinase (p42 aPk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42maPk on the regulatory tyrosine And to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42maPk. This p421aPk-activating activity is capable of phosphorylating a kinase-defective p42 lPk mutant, thus confirming its activity as a kinase.p42/microtubule-associated protein (MAP) kinase (p42maPk) is a serine/threonine protein kinase which rapidly becomes phosphorylated on threonine and tyrosine (42) in quiescent cells stimulated with various agonists, resulting in enzymatic activation. Both the threonine and tyrosine phosphorylations are necessary for full enzymatic activation (7,27,44), and the phosphorylation sites have been identified as T-183 and Y-185 (35).p42maPk is the paradigmatic member of an enzyme family which is activated by agonist stimulation in various cell types and species (for a recent review, see reference 12). Members of this family have also been termed MAP-2 kinases (23,27,(40)(41)(42), myelin basic protein (MBP) kinases (2, 4, 44) or extracellularly regulated kinases (ERKs) (9, 10). The p42maPk studied here corresponds to the MAP-2 kinase originally described by Ray and Sturgill (40), the MBP kinase 1 of Krebs et al. (2,4,44), the ERK2 of Boulton et al. (9,10), and the tyrosine phosphorylated protein pp42 described earlier by Cooper and Hunter (13,24) and by our (8,42) and other laboratories (17,26).Several candidate substrates for p42maPk and its family members have been identified, including MAP-2 (40), rsk ribosomal S6 kinase (11, 47), raf protein kinase (6), epidermal growth factor (EGF) receptor (35) Any one or a combination of three general mechanisms could be responsible for the regulatory phosphorylation of p42maPk: (i) a kinase cascade, in which an upstream kinase is regulated by agonists and phosphorylates p42m Pk, much as p42maPk phosphorylates rsk kinase (47); (ii) an intramolecular autokinase reaction, in which a nonkinase cellular factor regulates the ability of p42maPk to phosphorylate itself; (iii) an intermolecular autokinase reaction, in which p42maPk molecules are activated allosterically and then engage in mutual transphosphorylations, as occurs with many tyrosine kinase receptors.Previous work has demonstrated that p42"aPk is capable of slow intramolecular autophosphorylation (14, 46, 51) on tyrosine 185 (51), the site of in vivo regulatory phosphorylation on tyrosine. This reaction is accompanied by partial enzymatic activation. Because p42maPk is capable of this basal, regulatory autophosphorylation, we and others have speculated that the autokinase activity might be exploited in the regulatory phosphorylation which accom...

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk. Binding of the MPM-2 antibody to active p42mapk in vitro results in a decrease in p42mapk enzymatic activity. The MPM-2 phosphoepitope can be generated in vitro on bacterially expressed p42mapk by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42mapk proteins mutated in their regulatory sites shows that phosphorylated Thr-183 is essential for the binding of the MPM-2 antibody. MPM-2 binding to Thr-183 is affected by the amino acid present in the other regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalanine results in strong binding of the MPM-2 antibody, whereas substitution with glutamic acid substantially diminishes MPM-2 antibody binding. The MPM-2 phosphoepitope antibody recognizes an amino acid domain incorporating the regulatory phosphothreonine on activated p42mapk in eggs during meiosis and in mammalian cultured cells during the G0 to G1 transition.