MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

Abstract

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.

Signaling by dual specificity kinases

“…MKK1 and MKK2 in turn activate their respective ERKs through phosphorylation of speci®c Thr-and Tyr-residues within the characteristic TPY motif. The phosphorylated ERKs activate other signaling proteins such as cPLA 2 (Nemeno et al, 1993;Lin et al, 1993) as well as other kinases (Boulton et al, 1991;Sturgill et al, 1988;Stokoe et al, 1992). In addition, the phosphorylated ERKs translocate to nucleus (Chen et al, 1992;Seth et al, 1992;Sanghera et al, 1992;Lenormand et al, 1993) where they activate transcription factors such as TCFs through phosphorylation (Alvarez et al, 1991;Pulverer et al, 1991;Baker et al, 1992;Gille et al, 1992) which leads to the activation of speci®c genes involved in cell proliferation (Davis, 1992;Johnson and Vaillancourt, 1994).…”

Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo

“…The sequence identities of the peptides were also confirmed by Edman sequencing on an Applied Biosystems 476A sequenator. The sites of phosphorylation were determined by solid-phase Edman degradation of the peptide coupled to Sequelon-AA membrane (Milligen ; Bedford, MA, U.S.A.) as described previously [13]. Peptide P3 was sub-digested with 0.2 µg of Asp-N protease in 20 mM ammonium bicarbonate containing 0.1 % (w\w) n-octyl-glucoside (Calbiochem ; Nottingham, U.K.) for 6 h at 30 mC to yield a peptide termed D3.…”

“…In order to identify the site(s) of phosphorylation, an aliquot of each of the four major 32 P-labelled peptides, derived from 32 P-labelled PDK1 (Figure 1) of each peptide, was coupled covalently to a Sequelon arylamine membrane and analysed on an Applied Biosystems 470A sequencer using the modified programme [13]. 32 P-radioactivity was measured after each cycle of Edman degradation.…”

A comparison of the substrate specificity of MAPKAP kinase‐2 and MAPKAP kinase‐3 and their activation by cytokines and cellular stress

“…MAP kinase-activated protein kinase-2 (MAPKAP-K2) is a serine/threonine-specific protein kinase that is activated by p42 and p44 MAP kinases in vitro, but distinct from MAP-KAP kinase-1 (also known as p90 rsk) [1,2]. In vivo, however, MAPKAP-K2 is activated by a distinct MAP kinase homologue termed stress-activated protein kinase-2 (SAPK-2), also known as p38 [3], p40 [4], RK [5], CSBP [6] and Mxi2 [7].…”