Alexey Kotlyarov

MAPKAP kinase 2 is essential for LPS-induced TNF-|[alpha]| biosynthesis

MAPKAP kinase 2 (MK2) is one of several kinases that are regulated through direct phosphorylation by p38 MAP kinase. By introducing a targeted mutation into the mouse MK2 gene, we have determined the physiological function of MK2 in vivo. Mice that lack MK2 show increased stress resistance and survive LPS-induced endotoxic shock. This is due to a reduction of ∼90 % in the production of tumor necrosis factor-α (TNF-α) and not to a change in signalling from the TNF receptor. The level and stability of TNF-α mRNA is not reduced and TNF-α secretion is not affected. We conclude that MK2 is an essential component in the inflammatory response which regulates biosynthesis of TNF-α at a post-transcriptional level.

Regulation of Hsp27 Oligomerization, Chaperone Function, and Protective Activity against Oxidative Stress/Tumor Necrosis Factor α by Phosphorylation

The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.

Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology.

Interleukin‐10 (IL‐10) is a key inhibitory signal of inflammatory responses that regulates the production of potentially pathogenic cytokines like tumor necrosis factor (TNF). We show here that the development of chronic intestinal inflammation in IL‐10‐deficient mice requires the function of TNF, indicating that the IL‐10/TNF axis regulates mucosal immunity. We further show that IL‐10 targets the 3′ AU‐rich elements (ARE) of TNF mRNA to inhibit its translation. Moreover, IL‐10 does not alter TNF mRNA stability, and its action does not require the presence of the stability‐regulating ARE binding factor tristetraprolin, indicating a differential assembly of stability and translation determinants on the TNF ARE. Inhibition of TNF translation by IL‐10 is exerted mainly by inhibition of the activating p38/MAPK‐activated protein kinase‐2 pathway. These results demonstrate a physiologically significant cross‐talk between the IL‐10 receptor and the stress‐activated protein kinase modules targeting TNF mRNA translation. This cross‐talk is necessary for optimal TNF production and for the maintenance of immune homeostasis in the gut.

MAPKAP Kinase 2 Phosphorylates Serum Response Factor in Vitro and in Vivo

Several growth factor- and calcium-regulated kinases such as pp90 rsk or CaM kinase IV can phosphorylate the transcription factor serum response factor (SRF) at serine 103 (Ser-103). However, it is unknown whether stress-regulated kinases can also phosphorylate SRF. We show that treatment of cells with anisomycin, arsenite, sodium fluoride, or tetrafluoroaluminate induces phosphorylation of SRF at Ser-103 in both HeLa and NIH3T3 cells. This phosphorylation is dependent on the kinase p38/SAPK2 and correlates with the activation of MAPKAP kinase 2 (MK2). MK2 phosphorylates SRFin vitro at Ser-103 with similar efficiency as the small heat shock protein Hsp25 and significantly better than CREB. Comparison of wild type murine fibroblasts with those derived from MK2-deficient mice (Mk(−/−)) reveals MK2 as the major SRF kinase induced by arsenite. These results demonstrate that SRF is targeted by several signal transduction pathways within cells and establishes SRF as a nuclear target for MAPKAP kinase 2.

Protein Kinases as Substrates for SAPKs

The stress-activated protein kinases (SAPKs) consist of the c-Jun N-terminal kinases (JNKs or SAPK1αβγ) and of the four p38 MAPK-isoforms SAPK2a, b, SAPK3 and SAPK4 and phosphorylate a wide variety of different substrate proteins such as transcription factors, enzymes, and structural proteins. Of these kinases, only SAPK2s are known to phosphorylate and regulate downstream kinases; thereby, appending an additional level to this stress activated kinase cascade. Historically, these downstream kinases were designated as mitogen- and stress-activated protein kinases – MSKs, as MAPK-interacting protein kinases – MNKs, and as MAPK-activated protein kinases – MAPKAPKs or MKs. Although their kinase domains are phylogenetically related and show some similarities in regulation, the downstream kinases regulate gene expression at different levels, which contribute to orchestration of the stress response.