A novel protein kinase, which was only active when phosphorylated by the mitogen‐activated protein kinase (MAP kinase), has been purified 85,000‐fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase‐2, was distinguished from S6 kinase‐II (MAPKAP kinase‐1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase‐2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase‐2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase‐2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase‐2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor‐stimulated protein kinase cascade bifurcates.

SummaryBound antibodies can modulate antigen processing but it is not clear to what extent this affects antigen presentation. Here we show that presentation of T cell determinants in tetanus toxin can be either enhanced or suppressed as a direct consequence of antibody modulation of antigen processing in human B lymphoblastoid cells. Remarkably, a single bound antibody or its Fab fragment can simuhaneously enhance the presentation of one T cell determinant by more than 10-fold while strongly suppressing the presentation of a different T cell determinant. Biochemical analysis demonstrates that both the suppressed and boosted determinants fall within an extended domain of antigen stabilized or "footprinted" by this antibody during proteolysis. These results demonstrate that bound antibodies can modulate the capture of peptides by class II major histocompatibility complex (MHC), thus manipulating the T ceU response towards or away from particular determinants. Altered processing of protein-protein complexes leading to enhanced loading of class II MHC and substantially lowered thresholds for T cell activation suggests a novel mechanism that might reveal "cryptic" self determinants.T he possibility that bound antibodies might influence antigen processing and as a consequence affect its outcome at the level of presentation to T cells, has often been raised. Earlier studies showed specific effects on T cell proliferation when particular antigen-reactive antibodies were present (1-4). At the biochemical level proteolysis of protein-antibody complexes generated persistent protein fragments not seen in the absence of antibody. Such effects have been observed during in vitro digestion of antigen-antibody complexes (5, 6) and after antigen uptake into antigen-specific human B lymphocytes (7). However no mechanistic connection has been made between antibody modulation of processing at the biochemical level and possible outcomes at the level of T cell presentation.Recently we showed that the human monoclonal antibody 11.3 which is known to alter the course of tetanus toxin processing in human B cells (7) could block the appearance of a specific T cell determinant (8). Interference with loading of this determinant (1174-1189) was seen when this antibody specificity was present either as an antigen receptor on clone 11.3 B cells or when taken up in "piggyback" fashion into other B cells or macrophages, thus demonstrating that soluble antibodies can impose dominant and systemic effects on antigen processing. We now report that this same antibody simultaneously boosts by more than 10-fold the loading of a second determinant found approximately 100 residues downstream in the tetanus toxin molecule. Further, biochemical analysis of the region of antigen stabilized by antibody during digestion shows that both the suppressed and boosted determinants fall within its "footprint" Materials and MethodsCell Clones, Antigens, and Antibodies. EBV-transformed tetanus toxin-specific B cell clones 11.3, 4.2, and 8.5 and autologous T cell clone...

Mitogen activated protein (MAP) kinases and their target ribosomal protein S6 (RSK) kinases have been recognized as shared components in the intracellular signaling pathways of many diverse cytokines. Recent studies have extended this protein kinase cascade by identifying the major activator of vertebrate MAP kinases as a serine/threonine/tyrosine-protein kinase called MEK, which is related to yeast mating factor-regulated protein kinases encoded by the STE7 and byr1 genes. MEK, in turn, may be activated following its phosphorylation on serine by either of the kinases encoded by proto-oncogenes raf1 or mos, as well as by p78mekk, which is related to the yeast STE11 and byr2 gene products. Isoforms of all of these protein kinases may specifically combine to assemble distinct modules for intracellular signal transmission. However, the fundamental architecture of these protein kinase cascades has been highly conserved during eukaryotic evolution.

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