Heat-shock or stress proteins (HSPs) are considered to play an essential role in protecting cells from stress and preparing them to survive new environmental challenges. This study investigates the induction kinetics of synthesis and accumulation of 70-kDa stress proteins in the soleus and extensor digitorum longus (EDL) muscles of the rat following exercise, as well as the isoform transitions that take place during the post-exercise period. Relative synthesis rates (referred to constitutively expressed stress protein HSP73) of the 70-kDa heat-shock proteins were greatly enhanced after a single bout of exercise in both muscles. They peaked early in the post-exercise period and returned to resting levels after approximately 5-6 h. The levels of the inducible stress protein HSP72 in the EDL rose only transiently following exercise, while its accumulation in the soleus was more continuous and stable. The amount of HSP73 increased only transiently in both muscle types after exercise. The constitutive expression of the stress protein HSP72 in the soleus muscle was much higher than in the EDL and other tissues, while that of HSP73 was relatively constant among tissues. Rat skeletal muscle HSP72 and HSP73 were made up of at least three isoforms of the same molecular mass and very close isoelectric points, although only one radiolabelled isoform was detected. The relative proportion of the most abundant isoforms of HSP72, isoforms 1 and 2, as well as their ratio (isoform 2hoform l), increased during the post-exercise period. Since isoform 2 of HSP72 partially disappeared after incubating soleus muscle extracts of exercised rats with alkaline phosphatase, these data indicate that phosphorylation of HSP72 is an early event in the stress response of skeletal muscle to exercise stress.Keywords: heat-shock protein ; exercise ; skeletal muscle ; protein phosphorylation.Cells from all organisms studied thus far experience a stress response when subjected to high temperatures or other environmental challenges. This response is characterized by the induction of the synthesis of a group of proteins known as heat-shock or stress proteins (for a review see [ l , 21). In mammalian cells, the most highly induced proteins of the cellular stress response are the components of the protein family of 70 kDa, a group of closely related proteins that includes HSP72, HSP73, GRP78 and GRP75. All of them share the common property of binding ATP but have a different subcellular distribution within the cell [3, 41. HSP72 and HSP73 have been located within the cytoplasm and nucleus ; HSP73 is constitutively expressed while HSP72 is usually synthesized in response to stress. GRP78 is located in the lumen of the endoplasmic reticulum where it is expressed constitutively at a basal level and is induced by various factors that stress this cellular compartment. GRP78 binds Correspondence to R. Manso, Departamento de Biologia Molecular, Centro de Biologia Molecular 'Severo Ochoa' , Universidad Aut6noma de Madrid, Canto Blanco, E-28049 Madrid, SpainAbbr...

Cot, initially identified as an oncogene in a truncated form, is a mitogen-activated protein kinase kinase kinase implicated in cellular activation and proliferation. Here, we show that this truncation of Cot results in a 10-fold increase in its overall kinase activity through two different mechanisms. Truncated Cot protein exhibits a lower turnover rate (half-life, 95 min) than wild-type Cot (half-life, 35 min). The degradation of wild-type and truncated Cot can be specifically inhibited by proteasome inhibitors in situ. The 20S proteasome also degrades wild-type Cot more efficiently than the truncated protein. Furthermore, the amino acid 435 to 457 region within the wild-type Cot COOH-terminal domain confers instability when transferred to the yellow fluorescent protein and targets this fusion protein to degradation via the proteasome. On the other hand, the kinase specific activity of wild-type Cot is 3.8-fold lower than that of truncated Cot, and it appears that the last 43 amino acids of the wild-type Cot COOH-terminal domain are those responsible for this inhibition of kinase activity. In conclusion, these data demonstrate that the oncogenic activity of truncated Cot is the result of its prolonged half-life and its higher kinase specific activity with respect to wild-type Cot.The Cot/tpl-2 gene encodes a mitogen-activated protein (MAP) kinase kinase kinase that is potentially capable of switching on several MAP kinase cascades, namely those leading to the activation of the MAP kinases ERK1/ERK2, JNK, p38␥, and ERK5 (13,20,25,38,47,53). These signal transduction pathways link Cot activity with the up-regulation of several transcription factors, such as AP