Inactivation of Dual-Specificity Phosphatases Is Involved in the Regulation of Extracellular Signal-Regulated Kinases by Heat Shock and Hsp72

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 are mitogen-activated protein (MAP) kinases, which are preferentially triggered by growth-promoting stimuli such as growth factors and oncogenes (16, 27). Activation of MAP kinases proceeds through phosphorylation of specific Tyr and Thr residues catalyzed by the MEK kinase family. MEK kinases, in turn, are activated via phosphorylation by members of the Raf kinase family (16). Activation of the ERK pathway was shown to enhance cell resistance to a variety of stressful treatments, including heat shock (HS), growth factor withdrawal, hypoxia, chemotherapeutic agents, etc. (3, 5, 30, 35). Survival and proliferation of many types of tumor cells often rely on growth factor-stimulated ERK signaling, and many human tumors harbor hyperactivated components of ERK pathway (34). The exact mechanisms of ERK-mediated cell survival have not been established, but it is clear that ERKs may target different components of the death pathway. For example, it was shown that inhibition of ERK signaling sensitizes HeLa cells to apoptosis mediated by death receptors, such as Fas-R, TRAIL-R, or TNF-R (12, 31). In this system ERK seemed to control apoptosis at or upstream of caspase 8. It was also reported that the ERK signaling pathway is involved in the phosphorylation and the subsequent inactivation of the proapoptotic Bcl-2 family member, Bad (28). In another case, permanent activation of ERK by overexpression of B-Raf conferred on fibroblasts resistance to growth factor withdrawal. In this case, ERK intervened late in the apoptotic pathway, downstream of cytochrome c release (3). Heat stress is a potent inducer of apoptosis and necrosis in many cell types (7). Heating of cells triggers numerous signaling events, which contribute to either cell death or survival. For example, HS causes activation of stress kinase c-Jun N-terminal kinase (JNK), which promotes cell death, and duration of JNK activity closely correlates with the extent of cell death (32, 33). Furthermore, inhibition of JNK pathway greatly increases survival of cells upon HS (33). Besides stimulating JNK, HS also stimulates ERKs and, whereas activation of JNK triggers apoptosis, activation of ERKs may provide protection of cells from HS-mediated cell death (35). Indeed, specific inhibition of HS-activated ERK dramatically enhanced sensitivity of NIH 3T3 cells or primary human fibroblasts, whereas overexpression of ERKs makes cells resistant to heat-induced death (8, 35). Another survival mechanism triggered by HS is induction of HS proteins (Hsps) (25). Many Hsps, including the major inducible protein Hsp72, play a critical role in protection of cellular polypeptides from stress-induced denaturation and in refolding of damaged proteins (4, 11). In addition, Hsps directly interfere with several components of the apoptotic machinery, thus enhancing cell protection. For example, Hsp72 was shown to inhibit activation of JNK by various stressful treatments (19). These different signaling pathways have different thresholds and different rates of activation, which all affect the outcome of stressful treatments. Therefore, it seems that whether cells die or survive after HS depends upon an intricate balance of opposing signaling events. The mechanism of JNK activation upon HS has recently been established. It was shown that HS activates JNK mostly through inhibition of JNK dephosphorylation, without significant stimulation of JNK-activating kinase cascade (19). In unstressed cells, the kinase cascade has certain background activity, and JNK phosphorylation is kept at a very low level by highly active JNK phosphatases. After heat treatment the rate of JNK dephosphorylation is dramatically reduced, thus leading to stabilization of phosphorylated forms of JNK and the enhanced activity (19). Interestingly, Hsp72 inhibits HS-mediated activation of JNK through stimulation of JNK dephosphorylation (19). It was reported that brain-specific JNK phosphatase M3/6 dissociates from JNK6 after HS and becomes inactive and insoluble (23). Inactivation and aggregation of M3/6 appears to be the major event in activation of JNK by HS and potentially other protein-damaging stresses. It should be noted that in tissues other than brain distinct JNK phosphatases must be involved in HS regulation. In contrast to JNK, the mechanism of ERK activation upon HS is poorly understood. It was reported that in NIH 3T3 cells HS leads to activation of Raf1 through ceramide production (35). On the other hand, because of the homology of the JNK and ERK signaling pathway, we suggested that inhibition of ERK dephosphorylation might play a significant role in HS regulation. There are number of phosphatases that can dephosphorylate ERKs both in vitro and in vivo (26). For example, the serine/threonine-specific phosphatase PP2A can inactivate both MEK and ERK (1, 20). PP2A inhibition leads to hyperactivation of ERKs and cell transformation. Similarly, mutations in MEK, which lead to constitutive activation of ERK2, are accompanied by cellular transformation in fibroblasts (20). Another phosphatase involved in ERK signaling is MKP-1. MKP-1 is a dual specificity phosphatase induced by many stresses which activate ERKs (14). MKP-1 resides in the nucleus and is likely to be responsible for inactivation of ERKs upon their translocation into the nucleus. Yet another dual-specificity ERK phosphatase, MKP-3, is cytoplasmic and is expressed constitutively (10). MKP-3 binds ERK, and this binding renders MKP-3 catalytically active (2). In vivo MKP-3's specificity toward ERKs is very high, and MKP-3 was suggested to be the major phosphatase that keeps ERK activity at low levels in unstimulated cells. We investigated here the mechanisms of activation of ERKs by HS and the involvement of Hsp72and MKP-3 in this regulation.