7. Cyclic AMP and activation of CREB

26.6 Activating MAP kinase pathways

Key terms defined in this section
Scaffold of a chromosome is a proteinaceous structure in the shape of a sister chromatid pair, generated when chromosomes are depleted of histones.

Signal transduction generates differential responses to stimuli that vary qualitatively (by activating different pathways) or quantitatively (by activating pathways with different intensities or for different durations). An individual stimulus may activate one or more pathways. The strength of activation of any particular pathway may influence the response, since there are cases in which more intense or long-term stimulation of a single pathway gives a different response from less intense or short-term stimulation. One of our major aims is to understand how differences in such stimuli are transduced into the typical cellular responses.




Figure 26.20 A common signal transduction cascade passes from a receptor tyrosine kinase through an adaptor to activate Ras, which triggers a series of Ser/Thr phosphorylation events. Finally, activated MAP kinases enter the nucleus and phosphorylate transcription factors. Missing components are indicated by successive arrows.

One of the important features of signal transduction pathways is that they both diverge and converge, thus allowing different but overlapping responses to be triggered in different circumstances. Divergence may start with the initiating event. Activation of a receptor tyrosine kinase may itself trigger multiple pathways: for example, activation of EGF receptor activates the Ras pathway and also Ras-independent pathways involving second messengers (see Figure 26.20). There may also be "branches" later in a pathway (Lange-Carter et al., 1993).




Figure 26.19 Autophosphorylation triggers the kinase activity of the cytoplasmic domain of a receptor. The target protein may be recognized by an SH2 domain. The signal may subsequently be passed along a cascade of kinases.


Figure 26.21 The Ras cascade is initiated by a series of activation events that occur on the cytoplasmic face of the plasma membrane.

Convergence of pathways is illustrated by the ability of different types of initiating signal to lead to the activation of MAP kinases. The original paradigm and best characterized example of a pathway leading to MAP kinase involves the activation of Ras, as summarized in Figure 26.19. Returning to the early events in this pathway, the next component after Ras is the Ser/Thr (cytosolic) kinase, Raf. The relationship between Ras and Raf has been puzzling. We know that Ras and Raf are on the same pathway, because both of them are required for the phosphorylation of the proteins later in the pathway (such as MAP kinase). Ras must be upstream of Raf because it is required for the activation of Raf in response to extracellular ligands. Similarly, Raf must be downstream of Ras because the pathway triggered by Ras can be suppressed by expression of a dominant-negative (kinase deficient) mutant of Raf. Ras is localized on the cytoplasmic side of the plasma membrane, and its activation results in binding of Raf, which as a result is itself brought to the vicinity of the plasma membrane. However, the events that then activate Raf, and in particular the kinase that phosphorylates it, are not yet known. The present model is that Ras activates Raf indirectly, perhaps because some kinase associated with the membrane is constitutively active (see Figure 26.21). The importance of localization of enzymatic activities is emphasized by the abilities of components both upstream and downstream of Ras (that is, SOS and Raf) to exercise their activating functions as a consequence of being brought from the cytosol to the plasma membrane (Vojtek et al., 1993; Leevers et al., 1994).


Raf activity leads to the activation of MEK. Raf directly phosphorylates MEK, which is activated by phosphorylation on two serine residues. MEK is an unusual enzyme with dual specificity, which can phosphorylate both threonine and tyrosine. Its target is the ERK MAP kinase (Howe et al., 1992).


Both types of phosphorylation are necessary to convert a MAP kinase into the active state. There are at least 3 MAP kinase families, and they provide important switching points in their pathways. They are activated in response to a wide variety of stimuli, including stimulation of cell growth, differentiation, etc., and appear to play central roles in controlling changes in cell phenotype. The MAP kinases are serine/threonine kinases. After this point in the pathway, all the activating events take the form of serine/threonine phosphorylations.




Figure 26.27 A signal transduction cascade passes to the nucleus by translocation of a component of the pathway or of a transcription factor. The factor may translocate directly as a result of phosphorylation or may be released when an inhibitor is phosphorylated.

The ultimate effect of the MAP kinase pathway is a change in the pattern of transcription. So the initiating event occurs at the cell surface, but the final readout occurs in the nucleus, where transcription factors are activated (or inactivated). This type of response requires a nuclear localization step. General possibilities for this step are illustrated in Figure 26.27. In the classic MAP kinase pathway, it is accomplished by the movement of a MAP kinase itself to the nucleus, where it phosphorylates target transcription factors. An alternative pathway is to phosphorylate a cytoplasmic factor; this may be a transcription factor that then moves to the nucleus or a protein that regulates a transcription factor (for example, by releasing it to go to the nucleus).


The MAP kinases have several targets, including other kinases, such as Rsk, which extend the cascade along various branches. The ability of some MAP kinases to translocate into the nucleus after activation extends the range of substrates. In the classic pathway, ERK1 and ERK2 are the targets of MEK, and ERK2 translocates into the nucleus after phosphorylation. The direct end of one branch of the cascade is provided by the phosphorylation of transcription factors, including, c-Myc and Elk-1 (which cooperates with SRF [serum response factor]). This enables the cascade to regulate the activity of a wide variety of genes. (The important transcription factor c-Jun is phosphorylated by another MAPK, called JNK; see later (Wood et al., 1992; for review see Hill and Treisman, 1995).




Figure 26.28 Pathways activated by receptor tyrosine kinases and by serpentine receptors converge upon MEK.

In the MAP kinase pathway, MEK provides a convergence point. Ras activates Raf, which in turn activates MEK. Another kinase that can activate MEK is MEKK (MEK kinase), which is activated by G proteins, as illustrated in Figure 26.28. (We have not identified the component(s) that link the activated G protein to the MEKK.) So two principal types of stimulus at the cell surface Xactivation of receptor tyrosine kinases or of trimeric G proteins Xboth can activate the MAP kinase cascade. Formally, Raf and MEKK provide analogous functions in parallel pathways.




Figure 26.29 Homologous proteins are found in signal transduction cascades in a wide variety of organisms.

The counterparts for the components of the pathways in several organisms are summarized in Figure 26.29.


In mammals, fly, and worm, it starts by the activation of a receptor tyrosine kinase; in mammals the ligand is a polypeptide growth factor, in D. melanogaster retina it is a surface transmembrane protein on an adjacent cell (a "counter-receptor"), and in C. elegans vulval induction it is not known. The pathway continues through Grb2 in mammals, and through close homologs in the worm and fly. At the next stage, a homologue of SOS functions in the fly in the same way as in mammals. The pathway continues through Ras-like proteins (that is, monomeric guanine nucleotide-binding proteins) in all three higher eukaryotes. Mutations in a homologue of GAP also may influence the pathway in D. melanogaster, suggesting that there are alternative regulatory circuits, at least in flies. An interesting feature is that, although the Ras-dependent pathway is utilized in a variety of cells, the mutations in the SOS and GAP functions in Drosophila are specific for eye development; this implies that a common pathway may be regulated by components that are tissue-specific. There is a high degree of conservation of function; for example, Grb2 can substitute for Sem-5 in worms (Aroian et al., 1990; Hafen et al., 1987).


In yeast, the initiating event consists of the interaction of a polypeptide mating factor with a trimeric G protein, whose βγ dimer (STE2,3) activates the kinase STE20, which activates the MEKK, STE11. We do not know whether there are other components in addition to STE20 between Gβ γ and STE11, but the yeast pathway at present provides the best characterization of the route from a G protein to the MAP kinase cascade. The pathway then continues through components all of which have direct counterparts in yeast and mammals. STE7 is homologous to MEK, and FUS3 and KSS1 code for kinases that share with MAP kinase the requirement for activation by phosphorylation on both threonine and tyrosine. Their targets in turn directly execute the consequences of the cascade.


The MAP kinase cascade shown in Figure 26.29 is the best characterized, but there are also other, parallel cascades with related components. In yeast, in addition to the mating response pathway, cascades containing kinases homologous to MEKK, MEK, and MAPK respond to signaling initiated by changes in osmolarity, or activation of PKC (protein kinase C) in S. cerevisiae (for review see Herskowitz, 1995).




Figure 26.30 STE5 provides a scaffold that is necessary for MEKK, MEK, and MAPK to assemble into an active complex.

How is specificity established in the cascade: what prevents a component of one MAP kinase cascade activating the enzyme that corresponds to the next stage in a parallel pathway? One possible explanation is that the components may be localized. STE5 in yeast is implicated in the cascade between STE2,3 and FUS3, KSS1, but does not place in a single position. Figure 26.30 shows that STE5 binds to three of the kinases, STE11 (MEKK), STE7 (MEK), and FUS3 (MAPK), suggesting that this complex has to form before each kinase can activate the next kinase in the pathway. Each of the kinases binds to a different region on STE5, which provides a scaffold. If the kinases can only function in the context of the scaffold, they may be prevented from acting upon kinases in other pathways, and therefore act only upon one another (Choi et al., 1994).


What degree of amplification is achieved through the Ras/MAPK pathway? Typically an ~10 amplification of signal can be achieved at each stage of a kinase cascade, allowing an overall amplification of >104 through the pathway. However, the combination of the last three kinases into one complex would presumably restrict amplification at these stages. In mammalian cells, the pathway can be fully activated by very weak signals; for example, the ERK1,2 MAP kinases are fully activated when <5% of the Raf protein molecules bind to Ras.




Figure 26.31 JNK is a MAP-like kinase that can be activated by UV light or via Ras.

Another example of a pathway that proceeds through a MAP kinase is provided by the activation of the transcription factor Jun in response to stress signals. Figure 26.31 shows that activation of the kinase JNK involves both convergence and divergence. JNK is regulated by two classes of extracellular signals: UV light (typical of a stress response); and also as a consequence of activation of Ras (by an unidentified branch of the Ras pathway). JNK is a (distant) relative of MAP kinases such as the ERKs, showing the classic features of being activated by phosphorylation of Thr and Tyr, and phosphorylating its targets on Ser. The proteins JIP1,2 provide a scaffold that may ensure the integrity of the pathway leading to JNK activation (Derijard et al., 1994).




Figure 26.32 Three MAP kinase pathways have analogous components. Crosstalk between the pathways is shown by grey arrows.

The presence of multiple MAPK signaling pathways with analogous components is common. Figure 26.32 summarizes the mammalian pathways. Each pathway functions in a linear manner, as indicated previously, but in addition there may be "crosstalk" between the pathways, when a component in one pathway can activate the subsequent component in other pathways as well as its own. Usually these "lateral" signals are weaker than those propagating down the pathway. At the very start of the pathway, there is also signaling from Ras to Rac. The strengths of these lateral signals, as well as the extent of activation of an individual pathway, may be important in determining the biological response.


A puzzling feature of the Ras/MAPK pathway is that activation of the same pathway under different circumstances can cause different outcomes. When PC12 cells are treated with the growth factor NGF, they differentiate (by becoming neuronal-like) and stop dividing. When they are treated with EGF, however, they receive a signal for continued proliferation. In both cases, the principal signal transduction event is the activation of the ERK MAP kinase pathway. The differences in outcome might be explained, of course, by other (unidentified) pathways that are activated by the respective receptors. However, the major difference in the two situations is that NGF stimulation causes prolonged elevation of Ras-GTP, whereas EGF stimulation produces only a transient effect. (One reason for this difference is that EGF receptor is more susceptible to feedback mechanisms that reverse its activation.)


The idea that duration of the stimulus to the ERK MAPK pathway may be the critical parameter is supported by results showing that a variety of conditions that cause persistent activation of ERK MAP kinase, including constitutive activation of the EGF receptor, all cause differentiation. By contrast, all conditions in which activation is transient lead instead to proliferation. More direct proof of the role of the ERK MAPK pathway is provided by showing that mutations constitutively activating MEK cause differentiation of PC12 cells. So activation of the ERK MAPK pathway is sufficient to trigger the differentiation response. Another point is made by the fact that the same MEK mutation has different effects in a different host cell; in fibroblasts, it stimulates proliferation. This is another example of the ability of a cell to connect the same signal transduction pathway to different readouts (for review see Marshall, 1995).


How might the duration of the signal determine the type of outcome? The concentration of some active component in the pathway could increase with the duration of activation, and at some point would exceed a threshold at which it triggered a new response. One model for such an action is suggested by Drosophila development, in which increasing concentrations of a transcription factor activate different target genes, as the result of combinatorial associations with other factors that depend upon relative concentrations (see 29 Gradients, cascades, and signaling pathways). Another possibility is suggested by the fact that prolonged activation is required before ERK2 translocates to the nucleus. The mechanism is unknown, but could mean that transient stimulation does not support the phosphorylation and activation of nuclear transcription factors, so the expression of new functions (such as those needed for differentiation) could depend upon the stimulus lasting long enough to cause translocation of ERK2 (Cowley et al., 1994).




Reviews
Herskowitz, I. (1995). MAP kinase pathways in yeast: for mating and more. Cell 80, 187-198.
Hill, C. S. and Treisman, R. (1995). Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80, 199-212.
Marshall, C. J. (1995). Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179-186.

Research
Aroian, R. V. et al. (1990). The let-23 gene necessary for C. elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily. Nature 348, 693-699.
Choi, K.-Y. et al. (1994). Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell 78, 499-512.
Cowley, S. et al. (1994). Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH-3T3 cells. Cell 77, 841-862.
Derijard, B. et al. (1994). JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76, 1025-1037.
Hafen, E. et al. (1987). Sevenless, a cell-specific homeotic gene of Drosophila, encodes a putative transmembrane receptor with a tyrosine kinase domain. Science 236, 55-63.
Howe, L. R., Leevers, S. J., Gomez, N., Nakielny, S., Cohen, P., and Marshall, C. J. (1992). Activation of the MAP kinase pathway by the protein kinase raf. Cell 71, 335-342.
Lange-Carter, C. A. et al. (1993). A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science 260, 315-319.
Leevers, S. J., Paterson, H. F., and Marshall, C. J. (1994). Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369, 411-414.
Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993). Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74, 205-214.
Wood, K. W. et al. (1992). Ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell 68, 1041-1050.



Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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