Gsk3

Figure 6.3 Scheme of the mammalian MAPK signalling pathway, illustrating the great complexity of reactions. The figure shows a portion of a cell with the plasma and nuclear membranes. The classical pathway (on the right) starts with binding of a growth factor to an extracellular receptor (Y, receptor tyrosine kinase), leading to three successive waves of kinase activity (MAP3K, MAPKK, MAPK) and activation of MAPKAPs. When activated, several of the downstream kinases can translocate to the nucleus and trigger transcriptional regulation of a variety of genes. Kinases can also influence cytoplasmic targets and contribute to translational control. Parts of the MAPK pathway are activated by stress signals (on the left), but the cascade of events is less well known than that triggered by cell growth factors. ATF, activating transcription factor; GSK, glycogen synthase kinase; JAK, Janus kinase; PAK, p21-activated kinase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PLC, phospholipase C; S6K, ribosomal protein S6 kinase; SEK, SAPK kinase; STAT, signal transducer and activator of transcription. © Sigma—Aldrich Co.

receptor is occupied, a conformational change is imposed upon the intracellular domain, which then becomes an attractive binding site for adapter proteins, and their binding triggers further molecular reactions. The cascade of reactions following on RTK activation is extremely complex. More than 100 different proteins have been described to participate in the MAPK network; many of them are kinases, enzymes that activate other proteins by phosphorylating amino acids critical for the three-dimensional structure, resulting in activation or inactivation of the target. The MAPK cascade involves three successive tiers of kinase activity, which—from downstream to upstream—are designated MAPK, MAPK kinase (MAPKK, MKK, or MEK), and MAPKK kinase (MAPKKK, MAP3K, MEK kinase, or MEKK). The upstream proteins associated with binding to RTK and activation of MAP3K are sometimes called MAP4K (Fig. 6.3). Downstream of the three-tiered cascade, MAP kinases activate effector kinases, also called MAPK-activated proteins (MAPKAPs).

One way in which the MAPK proteins, and the effector kinases activated by them, exert their action is by translocation to the nucleus. An example is a kinase called extracellular signalregulated kinase (ERK), which when activated can pass through the nuclear envelope and there activate transcription factors. Important targets for ERK are c-Jun and c-Fos, which are components of the dimeric transcription factor activator protein 1 (AP-1), a protein which turns up as a downstream effector in various stress responses. The combination of two different proteins to a single active unity is called heterodimerization. Many of the transcription factors activated by MAPK are dimeric proteins, which only become active by combining the two components to one functional protein. Although transcription factors are important MAPK targets, MAPKs may also influence post-transcriptional processes in the cytoplasm, for example by contributing to mRNA stabilization.

MAPK is also activated by stress (Fig. 6.3). The part of the pathway that is specifically associated with the stress response is called the stress-activated protein kinase (SAPK) signalling pathway. However, the chain of events in SAPK is less well known than in the case of the classical pathway activated by mitogens. The stress signal may be transduced along a distinct pathway, interacting with the classical pathway, but it may also affect enzymes of the classical pathway directly. Two important kinases which are activated by stress and can translocate to the nucleus to activate transcription factors are HOG (product of high osmolarity gene) and c-Jun N-terminal kinase (JNK). Some of the MAPK proteins are known under different names in different organisms; for example HOG was first described in yeast but its orthologue in higher organisms is known as p38, whereas JNK is also known as SAPK5.

As noted above, the action of protein kinases involves phosphorylation of certain amino acids in other proteins. The two stress-related effector kinases, HOG and JNK, can only transfer to the nucleus when phosphorylated. However, they may also be dephosphorylated by the action of protein phosphatases. Phosphatases specific for MAPK are called MAPK phosphatases (MKPs;

Tamura et al. 2002). The action of these phospha-tases is very important for relaxation of the MAPK signal. There are four different MKP families; some of them act on specific effector kinases (e.g. only on JNK) and others have a broader action spectrum (e.g. they dephosphorylate three effector kinases— HOG, JNK, and ERK). Interestingly, MKP genes are among the many genes activated by MAPK signalling. Their activity thus constitutes a negative feedback, which attenuates the MAPK signal after being triggered by stress or a growth factor.

One of the possible ways in which stress may activate SAPK signalling is by inhibition of MKPs. Oxidative stress in particular may lead to the oxidation of sensitive thiol groups in protein phosphatases and so allow SAPK signalling by removing the negative feedback (Korsloot et al. 2004). Direct activation of kinases is another possible mechanism (Fig. 6.3).

The fact that different stimuli all activate MAPK signalling, yet can activate different genes, suggests the presence of a regulatory or coordinative system within the cascade. The existence of distinct but not mutually exclusive mechanisms, and the involvement of a large number of kinases, many of them acting upon each other, could contribute to the achievement of specificity in the responses to different stimuli.

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