In the p38 SAP kinase pathway, MAPKAP kinase-2 serves both as an effector of p38 by phosphorylating substrates and as a determinant of cellular localisation of p38. Nuclear export of p38 and MAPKAP kinase-2 may permit them to phosphorylate substrates in the cytoplasm.

MAP kinase‐activated protein (MAPKAP) kinase‐2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST‐MAPKAP kinase‐2 at Thr25 in the proline‐rich N‐terminal region Thr222 and Ser272 in the catalytic domain and Thr334 in the C‐terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase‐2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P‐labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SB 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P‐labelling of Thr338 is likely to result from autophosphorylation. GST‐MAPKAP kinase‐2 lacking the N‐terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full‐length GST‐MAPKAP kinase‐2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N‐terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase‐2, and this constitutively active form may be useful for studying its physiological roles.

It has recently been recognized that cellular stresses activate certain members of the mitogen-activated protein kinase (MAPK) superfamily. One role of these "stress-activated" MAPKs is to increase the transactivating activity of the transcription factors c-Jun, Elk1, and ATF2. These findings may be particularly relevant to hearts that have been exposed to pathological stresses. Using the isolated perfused rat heart, we show that global ischemia does not activate the 42- and 44-kD extracellular signal-regulated (protein) kinase (ERK) subfamily of MAPKs but rather stimulates a 38-kD activator of MAPK-activated protein kinase-2 (MAPKAPK2). This activation is maintained during reperfusion. The molecular characteristics of this protein kinase suggest that it is a member of the p38/reactivating kinase (RK) group of stress-activated MAPKs. In contrast, stress-activated MAPKs of the c-Jun N-terminal kinase (JNK/SAPKs) subfamily are not activated by ischemia alone but are activated by reperfusion following ischemia. Furthermore, transfection of ventricular myocytes with activated protein kinases (MEKK1 and SEK1) that may be involved in the upstream activation of JNK/ SAPKs induces increases in myocyte size and transcriptional changes typical of the hypertrophic response. We speculate that activation of multiple parallel MAPK pathways may be important in the responses of hearts to cellular stresses.

The Neu/HER‐2 receptor tyrosine kinase is overexpressed in some types of human adenocarcinomas, including tumors of the breast and the ovary. A 44 kDa glycoprotein that elevates tyrosine phosphorylation of Neu has been isolated and named Neu differentiation factor (NDF), or heregulin. Here we show that NDF affects tyrosine phosphorylation of Neu in human tumor cells of breast, colon and neuronal origin, but not in ovarian cells that overexpress the receptor. By using monoclonal antibodies (mAbs) to Neu, we found that the ovarian receptor is immunologically and biochemically similar to the mammary p185neu. Nevertheless, unlike breast‐derived Neu, the ovarian protein did not display covalent cross‐linking to radiolabeled NDF, and was devoid of ligand‐induced association with phosphatidylinositol 3′‐kinase. Direct binding analysis showed that NDF binds with high affinity (Kd approximately 10(−9) M) to mammary cells, but its weak association with ovarian cells is probably mediated by heparin‐like molecules. Similar to the endogenous receptor, the ectopically overexpressed Neu of mammary cells, but not of ovarian and fibroblastic cells, exhibited elevated levels of NDF‐induced phosphorylation and covalent cross‐linking of the radiolabeled factor. Taken together, our results imply that NDF binding to cells requires both Neu and an additional cellular component, whose identity is still unknown, but its tissue distribution is more restricted than the expression of the neu gene.

The transforming potential of the Neu/ErbB‐2 receptor tyrosine kinase undergoes inactivation by deletion of the non‐catalytic C‐terminal tail, which contains five autophosphorylation sites. To determine which site is essential for oncogenicity, we tailed the C‐terminally‐deleted mutant with individual autophosphorylation sites. Complete restoration of the transforming action in vitro and in vivo was conferred by a stretch of 12 amino acids that contained the most C‐terminal tyrosine autophosphorylation site (Y1253). Reconstitution of transformation was specific to this amino acid sequence because none of the other autophosphorylation sites, when grafted individually, caused transformation, and replacement of the tyrosine with a phenylalanine residue significantly reduced the oncogenic potential of both the full‐length and the tailed proteins. When present alone the most C‐terminal sequence enabled coupling to a biochemical pathway that includes Ras, MAP kinase and transactivation of Jun. These results indicate that the multiplicity of autophosphorylation sites on a receptor tyrosine kinase is not essential for transformability, and implicate the MAP kinase pathway in transduction of the oncogenic signal of Neu/ErbB‐2.