MAP klna~e.acttvatcd protein klna,e-= (MAPKAP klntts~.) phosphorylate, the sed,e t~.stdu~ in marine heat 1hock ~ ~(hlp~)m,lhuaumshock prot©ln is present in rabbit skeletal mu,¢le and hsl~$ kinue activity Ill skeletal mu~le extracts co~urtfln with MAPKAP klaaze~ actht~ throughout the purllicatton or the hitter enzyme. These p~sultm SUl;Sest that MAPKAP k|lla,t.-2 is the en~ me zeq~ondble f~r the photphoqlMim o1' these small heat shock protelm in mammalian ~elh.MAP klnase; Protein ktnase~ Heal shock~ Protein phosphorylatlon: Oro~lh factor i, INTRODUCTIONWe have recently identified a new protein kinase in rabbit skeletal muscle which is only active after it has been phosphorylatcd on a unique threonine residue by mitogen-activated protein kinase (MAP kinase) [1]. This enzyme, which has been termed MAP kinase activated ]2rotein kinase-2 (MAPKAP kinase-2) can be-distinguished from $6 kinase-II (or MAPKAP kinase-l) [2], the only other protein kinase known to be activated by MAP kinase, by its response to inhibitors, failure to phosphorylate peptides related to the C-temlinus of ribosomal protein $6 and by its amino acid sequence [1]. MAPKAP kinase-2 was originally identified by its ability to phosphorylat¢ rabbit skeletal muscle gl~.'ogen synthase, which it labels preferentially on a serine located seven residues from the N-terminus. It also phosphorylates the first serine in the peptide KKPLNRTLSVASLPGLamide, which is related to the N-terminus of glycogen synthase, and this substrate is used to assay MAPKAP kinase-2 routinely [I], Although glycogen synthase was the first substrate for MAPKAP kinase-2 to be identified, it is not clear whether it is phosphorylated by this protein kinase in vice, Furthermore, since MAPKAP kinase-i may phosphorylate more than one substrate in vice (e.g. the glycogen-bindin 8 subunit of protein phosphatase- ribosomal protein $6 [4]), MAPKAP kinme-2 may aim have a number of physiological substmtes and thereby mediate everal actions of extraodlular dSuab which exert their effects through the activation of MAP idmu~ and its downstream targets. We were lh¢~occ interested in identifying potential ph)~olosical subslrates for MAPKAP kinase-2.Murine heat shock protein ~ (hsp25) and its human homologue heat shock protein 27 (hsp27) are small thermostable proteins present in almost all nmmmalian cells, which become phosphorylated in many cells in response to signals such as tumour necrosis factor (TNF) [5,6], intedeuk,.'n-I [6,7], platelet derived growth factor (PDGF) [7] and fibroblast growth factor (FGF) [7,8], as well as turnout-promoting phorboi esters [9,10| and •heat shock [1 I--13], Their physiological roles are unknown, although overexpression of hsp27 and hsp25 has been reported to increase the thermotoleranceof some mammalian cells [14.15] and to inhibit cell proliferation [15|. Our interest in examining whether hsp25 and hsp27 x~re ph3-~ioiogical substrates for MAPKAP kinase-2 was amused by two observations. Firstly, several of the stimuli which trigger the phosphorylation of hsp25 a...

contributed equally to this work 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 Revtype 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.

Changes in environmental conditions such as the addition of growth factors or irradiation of cells in culture first affect immediate response genes. We have shown previously that short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor‐dependent pathways. At the level of the immediate response gene c‐fos, these pathways activate the transcription factor complex serum response factor (SRF)–p62TCF which mediates part of the UV‐induced transcriptional response. These studies have, however, suggested that more that one pathway is required for full UV responsiveness of c‐fos. Using appropriate promoter mutations and dominant‐negative cAMP response element (CRE)‐binding protein (CREB), we now find that UVC‐induced transcriptional activation depends also on the CRE at position −60 of the c‐fos promoter and on the functionality of a CREB. Upon UV irradiation, CREB and ATF‐1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG‐1 and of a p38/RK/HOG‐1‐dependent p108 CREB kinase. Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not, at least not decisively, participate in this signalling pathway to CREB and ATF‐1 as it is not p38/RK/HOG‐1 dependent, and CREB is a poor substrate for MAPKAP kinase 2 in vitro. On the basis of resistance to the growth factor receptor inhibitor suramin and of several types of cross‐refractoriness experiments, the UVC‐induced CREB/ATF‐1 phosphorylation represents an as yet unrecognized route of UVC‐induced signal transduction, independent of suramin‐inhibitable growth factor receptors and different from the Erk 1,2–p62TCF pathway.

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