The Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinases MK2 and MK3 Cooperate in Stimulation of Tumor Necrosis Factor Biosynthesis and Stabilization of p38 MAPK

Abstract

MK2 and MK3 represent protein kinases downstream of p38 mitogen-activated protein kinase (MAPK).Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, which displays extensive structural similarities and identical functional properties in vitro, is still present. Here, we analyze tumor necrosis factor (TNF) production and expression of p38 MAPK and tristetraprolin (TTP) in MK3-deficient mice and demonstrate that there are no significant differences with wild-type animals. We show that in vivo MK2 and MK3 are expressed and activated in parallel. However, the level of activity of MK2 is always significantly higher than that of MK3. Accordingly, we hypothesized that MK3 could have significant effects only in an MK2-free background and generated MK2/MK3 double-knockout mice. Unexpectedly, these mice are viable and show no obvious defects due to loss of compensation between MK2 and MK3. However, there is a further reduction of TNF production and expression of p38 and TTP in double-knockout mice compared to MK2-deficient mice. This finding, together with the observation that ectopically expressed MK3 can rescue MK2 deficiency similarly to MK2, indicates that both kinases share the same physiological function in vivo but are expressed to different levels.Downstream of mitogen-activated protein kinases (MAPKs) different groups of MAPK-activated protein kinases (MAP KAPKs) have been defined (reviewed in reference 28). These enzymes transduce signals to target proteins that are not direct substrates of the MAPKs and, therefore, serve to relay phosphorylation-dependent signaling within MAPK cascades to diverse cellular functions. One of these groups is formed by the three MAPKAPKs, MK2, MK3 (also known as 3pK), and MK5 (also designated PRAK) (reviewed in reference 12). While MK5 is mainly activated by the atypical MAPK ERK3 (29, 30), the remaining two kinases, MK2 and MK3, are directly downstream of the MAPK p38␣/␤ (7,10,24,27,31). Phosphorylation of MK2 and MK3 by p38␣/␤ at two or three major regulatory sites leads to activation and coupled nuclear export of both enzymes, which are localized in the nucleus of resting cells (4,8,26,36,41).A wide variety of substrates has been described for MK2 including proteins interacting with the cytoskeleton, such as small heat shock protein Hsp25 (33); mRNA-binding proteins, such as tristetraprolin (TTP) (6, 32); transcription factors, such as heat shock factor 1 (38); and regulators of the cell cycle and apoptosis, such as Cdc25B/C (23). The phosphorylation site recognition motifs of MK2 and MK3 are similar (20) or even identical (7). Despite the similar recognition motif, not all MK2 substrates have been described as MK3 substrates so far, probably because in most cells MK2 activity dominates and makes analysis of the minor MK3 activity dependent on antibodies which discriminate between both enzymes (7).MK2-deficient mice are more resistant than wild type to endotoxic shock due to impaired production of cytokines such as tumor necrosis factor (T...

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

“…The MAPKAP kinases MK2 and MK3 are highly similar in structure and function and, so far, no differences in their activation by p38 MAPK and their substrate specificity have been described [Clifton et al, 1996;Gaestel, 2006;Ronkina et al, 2007]. For MK2, a role in cell migration has been demonstrated which depends on its catalytic activity and on the integrity of its N-terminal proline-rich region [Kotlyarov et al, 2002].…”

“…The MK2-deficient or MK2/MK3 double-deficient immortalized mouse embryonic fibroblasts were transduced with the bicistronic pMMP-IRES MK2, MK2-K76R, and MK3 vectors following the previously reported method [Ronkina et al, 2007]. The different cell lines were sorted for comparable expression of GFP.…”

“…Since the migration behavior of immortalized MEFs can also be influenced by spontaneous mutations acquired in culture, we confirmed the involvement of MK2 in migration by a rescue experiment. Therefore, we transduced MK2-deficient MEFs with retroviral pMMP-IRES-GFP-constructs [Ronkina et al, 2007] coding for the expression of MK2 or GFP alone as a control. Expression of MK2, phospho-MK2 as well as rescue of p38 level and stress-induced Hsp25 phosphorylation in the transduced MEFs was monitored by Western blotting (Fig.…”

“…MK2 and MK3 are indistinguishable in regard to p38-dependent regulation and activation as well as substrate specificity [Clifton et al, 1996]. Both, MK2 and MK3, can rescue p38 level, Hsp25-phosphorylation, cytokine mRNA-stability, and CXCL1 production in MK2-deficient mouse embryonic fibroblasts [Ronkina et al, 2007].…”

Cullin 4B Is Recruited to Tristetraprolin-Containing Messenger Ribonucleoproteins and Regulates TNF-α mRNA Polysome Loading

“…TTP protein turnover is thought to be linked with TNF-a mRNA turnover (30,35,36); we thus examined the effect of Cul4B knockdown on TTP protein expression. Following LPS stimulation, TTP protein levels are reduced in Cul4B shRNA cells compared with control shRNA cells (Fig.…”