Katrin Engel

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.

Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation

To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein–MAPKAP kinase 2 fusion protein (GFP–MK2). In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm. This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation. Molecular mimicry of MK2 phosphorylation at T317 in GFP–MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress‐induced redistribution. Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev‐type leucine‐rich nuclear export signal (NES), blocks stress‐dependent translocation of GFP–MK2, it is supposed that phosphorylation‐induced export of the protein causes the translocation. We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C‐terminal to the autoinhibitory motif. This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine‐rich Rev‐type NES which is necessary to direct GFP–MK2 to the cytoplasm. However, unlike the Rev‐type NES, this region alone is not sufficient for nuclear export. The data obtained indicate that MK2 contains a constitutively active NLS and a stress‐regulated signal for nuclear export.

Constitutive activation of mitogen-activated protein kinase-activated protein kinase 2 by mutation of phosphorylation sites and an A-helix motif.

A recently described downstream target of mitogen-activated protein kinases (MAPKs) is the MAPK-activated protein (MAPKAP) kinase 2 which has been shown to be responsible for small heat shock protein phosphorylation. We have analyzed the mechanism of MAPKAP kinase 2 activation by MAPK phosphorylation using a recombinant MAPKAP kinase 2-fusion protein, p44 and p38/40in vitro and using an epitope-tagged MAPKAP kinase 2 in heat-shocked NIH 3T3 cells. It is demonstrated that, in addition to the known phosphorylation of the threonine residue carboxyl-terminal to the catalytic domain, Thr-317, activation of MAPKAP kinase 2 in vitro and in vivo is dependent on phosphorylation of a second threonine residue, Thr-205, which is located within the catalytic domain and which is highly conserved in several protein kinases. Constitutive activation of MAPKAP kinase 2 is obtained by replacement of both of these threonine residues by glutamic acid. A constitutively active form of MAPKAP kinase 2 is also obtained by deletion of a carboxyl-terminal region containing Thr-317 and the A-helix motif or by replacing the conserved residues of the A-helix. These data suggest a dual mechanism of MAPKAP kinase 2 activation by phosphorylation of Thr-205 inside the catalytic domain and by phosphorylation of Thr-317 outside the catalytic domain involving an autoinhibitory A-helix motif.

Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade

Hypo‐osmotic swelling of human Intestine 407 cells leads to a significant increase of intracellular MAPKAP‐kinase 2 activity and Hsp27 phosphorylation. Pre‐treatment of the cells with the p38 MAP kinase inhibitor SB‐203580 blocks this activation, indicating that the hypotonicity‐induced activation of MAPKAP kinase 2 is, similarly to that described for hyper‐osmotic treatment, the result of an activated p38 MAP kinase cascade. The activation of MAPKAP kinase 2 proceeds with kinetics similar to that of one of the first physiological responses of hypo‐osmotic treatment, the opening of compensatory Cl− channels. However, inhibition of the p38 MAP kinase cascade does not block the osmo‐sensitive anion efflux and, vice versa, activation of p38 MAP kinase by cytokines and anisomycin does not increase the efflux. These results indicate that the p38 MAP kinase cascade is not directly involved in Cl− channel activation but instead may play a role in subsequent cellular repair processes.

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.

The MAP kinase-activated protein kinase 2 contains a proline-rich SH3-binding domain.

The protein sequence of MAP kinase‐activated protein kinase 2 (MAPKAP kinase 2) deduced from mouse cDNA sequence reveals structural features of the enzyme, which could be of importance for its function: a proline‐rich SH3‐binding domain N‐terminal to the catalytic region, a MAP kinase phosphorylation site and a bipartite nuclear targeting sequence located C‐terminal to the catalytic region. The catalytic domain itself has the strongest homology to calcium/calmodulin‐dependent protein kinase II. Northern blot analysis demonstrates a 3.5 kb MAPKAP kinase 2 transcript which is ubiquitously expressed and, hence, co‐expressed with the mRNA of the recently identified substrate Hsp25 in all tissues analysed. However, the functional consequences of the nuclear targeting sequence present in MAPKAP kinase 2 suggest the existence of further substrates for the enzyme in the nucleus.

The protein kinase inhibitor SB203580 uncouples PMA-induced differentiation of HL-60 cells from phosphorylation of Hsp27.

HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2 (MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.