Matthias Gaestel

Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins

MAP kinase‐activated protein kinase‐2 (MAPKAP kinase‐2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser40 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Se86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co‐purifies with MAPKAP kinase‐2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase‐2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.

Over-expression of the small heat-shock protein, hsp25, inhibits growth of Ehrlich ascites tumor cells

hsp25 is a small, growth‐related, mammalian stress protein which is highly accummulated in the stationary phase or Ehrlich ascites tumor in vivo. Ehrlich ascites cells cultivated in vitro under conditions of continuous exponential growth express hsp25 only at a low level. These cells were stably transfected with an eukaryotic expression vector carrying the coding sequence of the small heat‐shock protein, hsp25, under control of the murine metallothionein promoter. The resulting cell lines (EAT 116 and EAT 118) exhibit constitutive over‐expression of the small heat‐shock protein. hsp25, which can be further increased by induction with cadmium. Both cell lines show increased thermoresistance. The in vitro proliferation rate of the transfected cell lines EAT 116 and EAT 118 is significantly decreased depending on the degree of cadmium‐regulated over‐expression of hsp25. Furthermore, a significant delay in Ehrlich ascites tumor growth in mice using the hsp25 over‐expressing cells for primary inoculation could be demonstrated.

PMA-induced activation of the p42/44ERK- and p38RK-MAP kinase cascades in HL-60 cells is PKC dependent but not essential for differentiation to the macrophage-like phenotype

The signaling mechanisms leading to phorbol ester myristate (PMA)‐induced differentiation of HL‐60 cells to the macrophagelike phenotype were investigated by using different protein kinase inhibitors. The protein kinase C inhibitor Ro 31‐8220 specifically blocks PMA‐induced differentiation, activation of the p42/44ERK‐ and p38RK‐MAP kinase cascades and Hsp27‐phosphorylation in HL‐60 cells. Because Ro 31‐8220 does not inhibit activation of the MAP kinase cascades by protein kinase C (PKC)‐independent signals such as epidermal growth factor (EGF), heat shock, or anisomycin in these cells, only PMA‐induced activation of the MAP kinases can be downstream of PKC. The MEK1 inhibitor PD 098059 and the p38RK inhibitor SB 203580 also were used to analyze whether the PMA‐induced PKC‐dependent activation of MAP kinases is involved in the differentiation process. Under certain conditions, PD 098059 can completely block the PMA‐induced activation of the p42ERK as monitored by imunoprecipitation kinase assay by using the substrate myelin basic protein. SB 203580 specifically inhibits activation of p38RK as judged by MAPKAP kinase 2 activity against the substrate Hsp27 and also blocks Hsp27 phosphorylation in the cells. In contrast, neither PD 098059 nor SB 203580 nor both inhibitors together prevent PMA‐induced differentiation of the HL‐60 cells to the macrophagelike phenotype. The results suggest the existence of a diversification of PMA‐induced signaling in HL‐60 cells downstream of PKC, leading to activation of MAP kinases that are not essential for differentiation and to phosphorylation of other, so far unidentified, targets responsible for differentiation. J. Cell. Physiol. 173:310–318, 1997.

Structure and organisation of a murine gene encoding small heat-shock protein Hsp25.

The structure and sequence of a gene encoding the mouse small heat-shock protein, Hsp25, is presented and compared to the human hsp27. In contrast to the human hsp27, only two copies of hsp25 could be detected in the mouse genome. The intron-exon structure of the identified hsp25 is similar to the transcribed human hsp27, and the transcription start points of the genes are located at similar sites. The promoter region contains various putative transcription factor-binding elements including two G + C-rich Sp1-binding domains, two heat-shock elements, and an estrogen-responsive element half-site in direct proximity to the TATA box. These elements could explain hsp25 basal expression as well as its induction as a result of heat-shock and estrogen treatment.

Dimer structure as a minimum cooperative subunit of small heat-shock proteins

Recently, it has been shown that small heat-shock proteins (Hsp25, Hsp27) are molecular chaperones. They bind to thermally unfolded proteins and can also assist refolding of denatured proteins. Mammalian small Hsps can form oligomeric structures of about 32 subunits. Until now, no data about cooperativity and stability of the interactions between the subunits of sHsps are available. To analyze these interactions we studied mouse Hsp25 and human Hsp27 by difference adiabatic scanning microcalorimetry (DASM) and circular dichroism (CD). Here we show that, according to DASM data, the minimum cooperatively melting structure is a sHsp-dimer. CD data indicate that Hsp25 major secondary structure, the beta-pleated conformation, is resistant to acidic influence up to pH 4.5 and, at neutral pH values, to heat treatment up to 60 degrees C. The melting pattern of Hsp25/27 bears resemblance to alpha-crystallins. CD data indicate similar secondary, tertiary and quaternary structures of the proteins compared. This finding is in agreement with the revealed homology of primary structure of these proteins and their common chaperone function.

The anti-cancer drug cisplatin induces HSP25 in Ehrlich ascites tumor cells by a mechanism different from transcriptional stimulation influencing predominantly HSP25 translation

Treatment of Ehrlich ascites tumor (EAT) cells with the anti‐cancer drug cisplatin induces an increase of the intracellular level of the small heat shock protein Hsp25 without stimulating the general stress response. The mechanism of this induction process was investigated at the levels of gene transcription, protein synthesis and stability. We show that an increased synthesis of Hsp25 is predominantly responsible for the increased intracellular level of this protein. In addition, there is a slightly increased metabolic stability of Hsp25 in cisplatin‐treated EAT cells. In contrast to the mechanism of Hsp25 induction by heat shock and other chemical stresses, stimulated synthesis of Hsp25 after treatment with cisplatin is not the result of increased transcription of the hsp25 gene. Cisplatin treatment does not significantly influence the oligomerization of heat shock transcription factors 1 and 2, hsp25 promoter activity or hsp25 mRNA stability, as judged by cross‐linking experiments, reporter gene assay and Northern blot analysis. Hence, cisplatin specifically induces Hsp25 synthesis at the level of mRNA translation without any changes in hsp25 gene transcription.

Blockade of MK2 is protective in inflammation-associated colorectal cancer development.

Chronic inflammation is a risk factor for colorectal cancer. The MAPK‐activated protein kinase 2 (MK2) pathway controls multiple cellular processes including p38‐dependent inflammation. This is the first study to investigate the role of MK2 in development of colitis‐associated colon cancer (CAC). Herein, we demonstrate that MK2−/− mice are highly resistant to neoplasm development when exposed to AOM/DSS, while wild type (WT) C57BL/6 develop multiple neoplasms with the same treatment. MK2‐specific cytokines IL‐1, IL‐6 and TNF‐α were substantially decreased in AOM/DSS treated MK2−/− mouse colon tissues compared with WT mice, which coincided with a marked decrease in macrophage influx. Restoring MK2‐competent macrophages by injecting WT bone marrow derived macrophages into MK2−/− mice led to partial restoration of inflammatory cytokine production with AOM/DSS treatment; however, macrophages were not sufficient to induce neoplasm development. These results indicate that MK2 functions as an inflammatory regulator to promote colonic neoplasm development and may be a potential target for CAC.