HspB1 is phosphorylated in the N-terminal part of the protein, and therefore outside of the alpha-crystallin domain, at serine sites 15, 78 and 82 by mitogen-activated protein kinases associated protein kinases 2,3. Similarly, HspB5 is phosphorylated at serines 19, 45 and 59. MAPKAPK2,3 phosphorylates serine 59 whereas serine 45 appears to be controlled by p42/p44 MAPKinase. The kinase responsive of serine 19 phosphorylation of HspB5 is still unknown. Phosphorylation is thought to act as a signaling mechanism regulating sHsps oligomerization since phosphomimetic mutants abolish, at least in cultured cells, HspB1 and HspB5 ability to oligomerize. This assumption is also supported by the fact that HspB1 amino terminus, which is involved in phosphorylation sensitive interactions, is crucial for oligomerization. Consequently, HspB1 and HspB5 holdase chaperone activity are regulated by the complex relationship that exists between their phosphorylation and oligomerization status. For example, it is particularly intriguing to note that in cells exposed to different environmental conditions or insults, HspB1 displays stress-specific changes in its oligomerization/phosphorylation status. Consequently, HspB1 probably acts as a protein sensor, which through structural changes, can interact with the most appropriate client protein targets. These phenomena subsequently allow cells to adapt to changes in their environment and/or mount a protective anti-stress response. In tissues that express several sHsps, such as in lens and ICG-001 muscles, these proteins can interact and form multiple combinatorial oligomeric structures that can bear different functions. One example is the 3 to 1 unique large chimeric oligomer formed by HspB4 and HspB5 in lens fiber cells. This oligomeric structure appears to have a higher stability and to be a more efficient chaperone than the individual polypeptides. Indeed, in spite of their high degree of homology, HspB4 and HspB5 polypeptides are characterized by their conformational and functional differences. HspB5 is, for example, more susceptible than HspB4 to heat-induced conformational change and aggregation. In tissues where HspB1 is expressed along with HspB5, it interacts with HspB5 and may serve, as HspB4 does in the lens, to chaperone and stabilize HspB5 conformation, particularly in stress conditions. Moreover, the subunit SCH772984 ERK inhibitor exchange between HspB5 and HspB1 is more rapid than between HspB5 and HspB4. The protective effect of HspB1 is particularly intense towards the R120G mutant of HspB5, an unstable polypeptide prone to aggregate. Of interest, HspB1 increases its chaperone-like activity by interacting with HspB5. The goal of this study was to examine stable HeLa cell clones that express similar levels of either wild type or R120G mutated HspB5 and endogenous HspB1. We show pronounced and opposite effects induced by wild type and mutant HspB5 on cell morphology and oxidoresistance. The R120G mutation increased the native size of HspB1-HspB5 complex and its resistance to saltinduced dissociation. It also allowed the phosphorylation of HspB1 serine15 in the complex, a modification that stabilized HspB1 interaction with mutant HspB5. In oxidative conditions, the partial dissociation of HspB1-HspB5 complex was drastically enhanced in cells expressing mutant HspB5, a phenomenon followed by the aggregation of the two protein partners. In addition to the chaperone effect of HspB1 towards mutant HspB5, these observations enlighten the major dominant positive and negative effects of HspB5 towards HspB1 in normal and oxidative conditions. Following transfection and selection, G418 resistant HeLa clones that express either wild type or R120G mutant HspB5 were analyzed in immunoblots. Comparison with the signals generated by serial dilutions of pure HspB5 revealed that two clones expressed similar levels of wild type or mutant HspB5.