Info

Inactive Raf

N-terminal regulatory domain

C-terminal kinase domain

▲ FIGURE 14-21 Kinase cascade that transmits signals downstream from activated Ras protein to MAP kinase. In unstimulated cells, most Ras Is In the Inactive form with bound GDP; binding of a ligand to Its RTK or cytokine receptor leads to formation of the active Ras-GTP complex (step 1 ; see also Figure 14-16). Activated Ras triggers the downstream kinase cascade depicted in steps 2 - 6, culminating in activation of MAP kinase (MAPK). In unstimulated cells, binding of the 14-3-3 protein to Raf stabilizes it in an inactive conformation. Interaction of the Raf N-terminal regulatory domain with Ras-GTP relieves this inhibition, results in dephosphorylation of one of the serines that bind Raf to 14-3-3, and leads to activation of Raf kinase activity (steps 2 and 3). Note that in contrast to many other protein kinases, activation of Raf does not depend on phosphorylation of the activation lip. After inactive Ras-GDP dissociates from Raf, it presumably can be reactivated by signals from activated receptors, thereby recruiting additional Raf molecules to the membrane. See the text for details. [See E. Kerkhoff and U. Rapp, 2001, Adv.: Enzyme Regul. 41:261; J. Avruch et al., 2001, Recent Prog. Hormone Res. 56:127; and M. Yip-Schneider et al., 2000, Biochem. J. 351:151

GTP hydrolysis leads to dissociation of Ras from Raf

MAP kinase

Dimeric form of active MAP kinase translocates to nucleus; activates many transcription factors

MAP kinase

Dimeric form of active MAP kinase translocates to nucleus; activates many transcription factors a link between the Raf and Ras proteins. In vitro binding studies further showed that the purified Ras-GTP complex binds directly to the N-terminal regulatory domain of Raf and activates its catalytic activity. An interaction between the mammalian Ras and Raf proteins also was demonstrated in the yeast two-hybrid system, a genetic system in yeast used to select cDNAs encoding proteins that bind to target, or "bait," proteins (see Figure 11-39).

That MAP kinase is activated in response to Ras activation was demonstrated in quiescent cultured cells expressing a constitutively active RasD protein. In these cells activated MAP kinase is generated in the absence of stimulation by growth-promoting hormones. More importantly, R7 pho-toreceptors develop normally in the developing eye of Drosophila mutants that lack a functional Ras or Raf protein but express a constitutively active MAP kinase. This finding indicates that activation of MAP kinase is sufficient to transmit a proliferation or differentiation signal normally initiated by ligand binding to a receptor tyrosine kinase such as Sev-enless (see Figure 14-18). Biochemical studies showed, however, that Raf cannot directly phosphorylate MAP kinase or otherwise activate its activity.

The final link in the kinase cascade activated by Ras-GTP emerged from studies in which scientists fractionated extracts of cultured cells searching for a kinase activity that could phosphorylate MAP kinase and that was present only in cells stimulated with growth factors, not quiescent cells. This work led to identification of MEK, a kinase that specifically phosphorylates one threonine and one tyrosine residue on MAP kinase, thereby activating its catalytic activity. (The acronym MEK comes from MAP and ERK kinase.) Later studies showed that MEK binds to the C-terminal catalytic domain of Raf and is phosphorylated by the Raf serine/ threonine kinase; this phosphorylation activates the catalytic activity of MEK. Hence, activation of Ras induces a kinase cascade that includes Raf, MEK, and MAP kinase: activated RTK ^ Ras ^ Raf ^ MEK ^ MAP kinase.

Activation of Raf Kinase The mechanism for activating Raf differs from that used to activate many other protein kinases including MEK and MAP kinase. In a resting cell prior to hormonal stimulation, Raf is present in the cytosol in a conformation in which the N-terminal regulatory domain is bound to the kinase domain, thereby inhibiting its activity. This inactive conformation is stabilized by a dimer of the 143-3 protein, which binds phosphoserine residues in a number of important signaling proteins. Each 14-3-3 monomer binds to a phosphoserine residue in Raf, one to phosphoserine-259 in the N-terminal domain and the other to phosphoserine-621 (see Figure 14-21). These interactions are thought to be essential for Raf to achieve a conformational state such that it can bind to activated Ras.

The binding of Ras-GTP, which is anchored to the membrane, to the N-terminal domain of Raf relieves the inhibition of Raf's kinase activity and also induces a confor-mational change in Raf that disrupts its association with

14-3-3. Raf phosphoserine-259 then is dephosphorylated (by an unknown phosphatase) and other serine or threonine residues on Raf become phosphorylated by yet other kinases. These reactions incrementally increase the Raf kinase activity by mechanisms that are not fully understood.

Activation of MAP Kinase Biochemical and x-ray crystallo-graphic studies have provided a detailed picture of how phosphorylation activates MAP kinase. As in JAK kinases and the cytosolic domain of receptor tyrosine kinases, the catalytic site in the inactive, unphosphorylated form of MAP kinase is blocked by a stretch of amino acids, the activation lip (Figure 14-22a). Binding of MEK to MAP kinase destabilizes the lip structure, resulting in exposure of tyrosine-185, which is buried in the inactive conformation. Following phosphorylation of this critical tyrosine, MEK phosphory-lates the neighboring threonine-183 (Figure 14-22b).

Both the phosphorylated tyrosine and the phosphory-lated threonine residues in MAP kinase interact with additional amino acids, thereby conferring an altered conformation to the lip region, which in turn permits binding of ATP to the catalytic site. The phosphotyrosine residue (pY185) also plays a key role in binding specific substrate proteins to the surface of MAP kinase. Phosphorylation promotes not only the catalytic activity of MAP kinase but also

(a) Inactive MAP kinase

(a) Inactive MAP kinase

(b) Active MAP kinase

(b) Active MAP kinase

▲ EXPERIMENTAL FIGURE 14-22 Molecular structures of MAP kinase in its inactive, unphosphorylated form (a) and active, phosphorylated form (b). Phosphorylation of MAP kinase by MEK at tyrosine-185 (Y185) and threonine-183 (T183) leads to a marked conformational change in the activation lip. This change promotes dimerization of MAP kinase and binding of its substrates, ATP and certain proteins. A similar phosphorylation-dependent mechanism activates JAK kinases, the intrinsic kinase activity of RTKs, and MEK. [After B. J. Canagarajah et al., 1997, Cell 90:859.]

its dimerization. The dimeric form of MAP kinase (but not the monomeric form) can be translocated to the nucleus, where it regulates the activity of many nuclear transcription factors.

MAP Kinase Regulates the Activity of Many Transcription Factors Controlling Early-Response Genes

Addition of a growth factor (e.g., EGF or PDGF) to quiescent cultured mammalian cells in G0 causes a rapid increase in the expression of as many as 100 different genes. These are called early-response genes because they are induced well before cells enter the S phase and replicate their DNA (see Figure 21-29). One important early-response gene encodes the transcription factor c-Fos. Together with other transcription factors, such as c-Jun, c-Fos induces expression of many genes encoding proteins necessary for cells to progress through the cell cycle. Most RTKs that bind growth factors utilize the MAP kinase pathway to activate genes encoding proteins like c-Fos that propel the cell through the cell cycle.

The enhancer that regulates the c-fos gene contains a serum-response element (SRE), so named because it is activated by many growth factors in serum. This complex enhancer contains DNA sequences that bind multiple transcription factors. Some of these are activated by MAP kinase, others by different protein kinases that function in other signaling pathways (e.g., protein kinase A in cAMP pathways and protein kinase C in phospho-inositide pathways).

As depicted in Figure 14-23, activated (phosphorylated) dimeric MAP kinase induces transcription of the c-fos gene by modifying two transcription factors, ternary complex factor (TCF) and serum response factor (SRF). In the cytosol, MAP kinase phosphorylates and activates another kinase, p90RSK, which translocates to the nucleus, where it phos-phorylates a specific serine in SRF. After also translocating to the nucleus, MAP kinase directly phosphorylates specific serines in TCF. Association of phosphorylated TCF with two molecules of phosphorylated SRF forms an active trimeric factor that binds strongly to the SRE DNA segment. As evidence for this model, abundant expression in cultured mammalian cells of a mutant dominant negative TCF that lacks the serine residues phosphorylated by MAP kinase blocks the ability of MAP kinase to activate gene expression driven by the SRE enhancer. Moreover, biochemical studies showed directly that phosphorylation of SRF by active p90RSK increases the rate and affinity of its binding to SRE sequences in DNA, accounting for the increase in the frequency of transcription initiation. Thus both transcription factors are required for maximal growth factor-induced stimulation of gene expression via the MAP kinase pathway, although only TCF is directly activated by MAP kinase.

Phosphorylation of transcription factors by MAP kinase can produce multiple effects on gene expression. For instance, two related Drosophila transcription factors, Pointed

Active, dimeric MAP kinase ATP

Active, dimeric MAP kinase ATP

c-fos gene

▲ FIGURE 14-23 Induction of gene transcription by activated MAP kinase. In the cytosol, MAP kinase phosphorylates and activates the kinase p90RSK, which then moves into the nucleus and phosphorylates the SRF transcription factor. After translocating into the nucleus, MAP kinase directly phosphorylates the transcription factor TCF Together, these phosphorylation events stimulate transcription of genes (e.g., c-fos) that contain an SRE sequence in their promoter. See the text for details. [See R. Marais et al., 1993, Cell 73:381, and V. M. Rivera et al., 1993, Mol. Cell Biol. 13:6260.]

and Yan, which are directly phosphorylated by MAP kinase, are crucial effectors of RTK signaling in the eye and other tissues. Phosphorylation enhances the activity of Pointed, a transcriptional activator. In contrast, unphosphorylated Yan is a transcriptional repressor that accumulates in the nucleus and inhibits development of R7 cells in the eye. Following signal-induced phosphorylation, Yan accumulates in the cy-tosol and does not have access to the genes it controls, thereby relieving their repression. Mutant forms of Yan that cannot be phosphorylated by MAP kinase are constitutive repressors of R7 development. This example suggests that a complex interplay among multiple transcription factors, regulated by signal-activated kinases, is critical to cellular development.

G Protein-Coupled Receptors Transmit Signals to MAP Kinase in Yeast Mating Pathways

Although many MAP kinase pathways are initiated by RTKs or cytokine receptors, signaling from other receptors can activate MAP kinase in different cell types of higher eukary-otes. Moreover, yeasts and other single-celled eukaryotes, which lack cytokine receptors or RTKs, do possess several MAP kinase pathways. To illustrate, we consider the mating pathway in S. cerevisiae, a well-studied example of a MAP kinase cascade linked to G protein-coupled receptors (GPCRs), in this case for two secreted peptide pheromones, the a and a factors.

As discussed in Chapter 22, these pheromones control mating between haploid yeast cells of the opposite mating type, a or a. An a haploid cell secretes the a mating factor and has cell-surface receptors for the a factor; an a cell secretes the a factor and has cell-surface receptors for the a factor (see Figure 22-13). Thus each type of cell recognizes the mating factor produced by the opposite type. Activation of the MAP kinase pathway by either the a or a receptors in duces transcription of genes that inhibit progression of the cell cycle and others that enable cells of opposite mating type to fuse together and ultimately form a diploid cell.

Ligand binding to either of the two yeast pheromone receptors triggers the exchange of GTP for GDP on the single Ga subunit and dissociation of Ga-GTP from the Gp7 complex. This activation process is identical to that for the GPCRs discussed in the previous chapter (see Figure 13-11). In most, but not all, mammalian GPCR-initiated pathways, the active Ga transduces the signal. In contrast, mutant studies have shown that the dissociated Gp7 complex mediates all the physiological responses induced by activation of the yeast pheromone receptors. For instance, in yeast cells that lack Ga, the Gp7 subunit is always free. Such cells can mate in the absence of mating factors; that is, the mating response is con-stitutively on. However, in cells defective for the Gp or G7 subunit, the mating pathway cannot be induced at all. If dissociated Ga were the transducer, the pathway would be expected to be constitutively active in these mutant cells.

In yeast mating pathways, Gp7 functions by triggering a kinase cascade that is analogous to the one downstream from

Exterior

Mating factor

Receptor

Mating factor

Receptor

Activation of G protein

Cytosol

Activation of G protein

Cytosol

Ste5

scaffold protein

Ste5

scaffold protein

Serine/ P threonine kinase

MEK,

P threonine/tyrosine dual-specificity kinase

MAPK, % serine/threonine kinase

Fus3 to nucleus

Serine/ P threonine kinase

MEKK, P serine/threonine kinase

MEK,

P threonine/tyrosine dual-specificity kinase

MAPK, % serine/threonine kinase

Fus3 to nucleus c

Ste 12 Transcription factor

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