Identification of Mitogen-activated Protein (MAP) Kinase-activated Protein Kinase-3, a Novel Substrate of CSBP p38 MAP Kinase

Abstract

CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares approximately 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.

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

“…4). The activation of an epitope-tagged version of MAPKAP-K3 by TNF or osmotic shock and blockade of its activation by SB 203580 has been demonstrated previously after its transfection into HeLa cells [18]. However, by raising antibodies that immunoprecipitate either MAPKAP-K2 or MAPKAP-K3, we have been able to study the activation of the endogenous enzymes in the same cell lysates.…”

“…MAPKAP-K2 purified from rabbit skeletal muscle was provided by [18]) was induced with 30 ~tM isopropyl-13-D-thiogalactoside for 20 h at 22°C. 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 protein kinase, termed MAPKAP-K3, has recently been identified using the yeast 'two hybrid' system to identify proteins that interact with SAPK-2 and shown to be activated by SAPK-2 in vitro [18]. DNA encoding the same protein kinase was also identified while sequencing a region of human chromosome 3 that is frequently deleted in small cell lung carcinomas [19].…”

Activation and signaling of the p38 MAP kinase pathway

“…The first p38α substrate identified was the MAP kinase-activated protein kinase 2 (MAPKAPK2 or MK2) [1,15,52]. This substrate, along with its closely related family member MK3 (3pk), were both shown to activate various substrates including small heat shock protein 27 (HSP27) [53], lymphocyte-specific protein 1 (LSP1) [54], cAMP response element-binding protein (CREB) [55], transcription factor ATF1 [55], SRF [56], and tyrosine hydroxylase [57].…”

Fluorescence‐based quantitative scratch wound healing assay demonstrating the role of MAPKAPK‐2/3 in fibroblast migration

“…p38a Mitogen-activated protein kinase (MAPK)-deficient [Rousseau et al, 2006] and MAP kinase-activated protein kinase 2 (MK2)-deficient fibroblasts showed reduced ability to migrate in scratch wound assays [Liu et al, 2007]. Apart from MK2, mammalian cells harbor a related p38-regulated protein kinase designated MK3 or 3pK [McLaughlin et al, 1996, Sithanandam et al, 1996. MK2 and MK3 show 75 % overall sequence identity on the amino acid level with the highest similarity within the catalytic domain and lower conservation in the N-terminal region [Gaestel, 2006], which displays a clear proline-rich motif in MK2 but not in MK3.…”