Autophosphorylation in vitro of recombinant 42-kilodalton mitogen-activated protein kinase on tyrosine.

Abstract

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...

Phosphoregulatory Tyrosine of Xenopus Mitogen‐Activated Protein Kinase is out of the Reach of the Enzyme Catalytic Center After Autophosphorylation

“…Even prior to the in vitro autophosphorylation, MAPK had some basal MBPphosphorylating activity which can be explained by the presence of the Tyr-phosphorylated form of enzyme in the original preparation of recombinant MAPK, as revealed by blotting with antiphosphotyrosine PY20 (data not shown). Phosphoamino acid analysis of in-vitro autophosphorylated Xenopus MAPK confirmed that Tyr is the major site of autophosphorylation with a minor substoichiometric phosphorylation of Ser, as reported earlier been demonstrated to be intramolecular (Robbins et al, 1993;Wu et al, 1991). In this study, the intramolecular character of Xenopus MAPK autophosphorylation on Tyr was confirmed in three kinds of experiments.…”

“…Thus, the single-site autophosphorylation on Tyr accounts for the observed activation of Xenopus MAPK. Significant (25-45 %) Tyr phosphorylation of mammalian MAPK during the bacterial expression has been reported (Wu et al, 1991). However, our preparation of Xenopus MAPK contained only about 0.5 % of the initially Tyr-phosphorylated enzyme (data not shown), which made it possible to register activation upon autophosphorylation without the separation of phosphorylated and unphosphorylated forms (Fig.…”

“…Autophosphorylation also leads to the partial activation of the enzyme, although the magnitude of this process is considerably lower than in the case of MAPKK action (Seger et al, 1991 ;Wu et al, 1991). The major autophosphorylation site of mammalian MAPKs (ERK1 and ERK2) is Tyr185 (Rossomando et al, 1992), however, phosphate incorporation into unidentified Thr (Seger et al, 1991 ;Wu et al, 1991) and into Thr and Ser (Robbins et al, 1993) has also been detected.…”

“…The major autophosphorylation site of mammalian MAPKs (ERK1 and ERK2) is Tyr185 (Rossomando et al, 1992), however, phosphate incorporation into unidentified Thr (Seger et al, 1991 ;Wu et al, 1991) and into Thr and Ser (Robbins et al, 1993) has also been detected. In the case of Xenopus MAPK,which has 97% similarity with ERK2 (Gotoh et al, 1991 b), the major phosphorylation on Tyr accompanied by a minor Ser phosphorylation has been reported .…”

“…MAPK is capable of intramolecular autophosphorylation, which is rather slow compared to MAPKK-mediated phosphorylation and is not considered to play an important physiological role (Seger et al, 1991 ;Wu et al, 1991 ;Robbins et al, 1993).…”