Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2.

Abstract

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.

Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS

“…Mutation of the equivalent site in MNK1 or MNK2 has also been reported to give similar results [31,32]; however, while this site is phosphorylated in vitro by ERK [33], it was unclear from these studies if this was an in vivo phosphorylation site. Interestingly the equivalent site (Thr 338 ) is phosphorylated in MAPKAPK2, although this probably occurs via autophosphorylation rather than by p38 or ERK1/2 [34]. A MAPK site (Thr 334 ) does exist in MAPKAPK2 four residues N-terminal to the Thr 338 autophosphorylation site.…”

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

“…The glutathione-S-transferase (GST)-MAPKAP-K3 fusion protein was purified by affinity chromatography on glutathione-Sepharose (Sigma) and showed two bands migrating on SDS/polyacrylamide gels with apparent molecular masses of 67 and 65 kDa. A fusion protein consisting of GST followed by residues 46-400 of MAPKAP-K2 was expressed in E. coli and purified as described [11]. This preparation also migrates as two bands with apparent molecular masses of 65 and 62 kDa.…”

“…Each enzyme was activated by incubation with the Xenopus homologue of SAPK-2 [11] which had been expressed in E. coli [5] and then activated by the MAP kinase kinase homologue MKK6 from skeletal muscle [20]. Only the 65 kDa band of MAPKAP-K2 [11] and the 67 kDa band of MAPKAP-K3 are phosphorylated by SAPK-2. Activated MAPKAP-K2 and MAPKAP-K3 were mixed with equal volumes of glycerol and stored unfrozen at -20°C.…”

“…The activation of MAP-KAP-K2 by SAPK-2 in vitro results from the phosphorylation of Thr-222 and Ser-272 in the catalytic domain and Thr-334 in the C-terminal tail. Phosphorylation of any two of these three residues is sufficient for maximal activation [11]. These residues also become phosphorylated in vivo in an SB 203508 sensitive manner when KB cells are stimulated with sodium arsenite [11].…”

“…Phosphorylation of any two of these three residues is sufficient for maximal activation [11]. These residues also become phosphorylated in vivo in an SB 203508 sensitive manner when KB cells are stimulated with sodium arsenite [11]. *Corresponding author.…”