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Genetic Studies with S. pombe

The studies with Xenopus egg extracts described in the previous section showed that continuous synthesis of a mitotic cyclin followed by its periodic degradation at late anaphase is required for the rapid cycles of mitosis observed in early Xenopus embryos. Identification of the catalytic protein ki-nase subunit of MPF and further insight into its regulation came from genetic analysis of the cell cycle in the fission yeast ,S. pombe. This yeast grows as a rod-shaped cell that increases in length as it grows and then divides in the middle during mitosis to produce two daughter cells of equal size (Figure 21-11).

In wild-type ,S. pombe, entry into mitosis is carefully regulated in order to properly coordinate cell division with cell growth. Temperature-sensitive mutants of S. pombe with conditional defects in the ability to progress through the cell cycle are easily recognized because they cause characteristic changes in cell length at the nonpermissive temperature. The many such mutants that have been isolated fall into two groups. In the first group are cdc mutants, which fail to progress through one of the phases of the cell cycle at the nonpermissive temperature; they form extremely long cells because they continue to grow in length, but fail to divide. In contrast, wee mutants form smaller-than-normal cells because they are defective in the proteins that normally prevent cells from dividing when they are too small.

In S. pombe wild-type genes are indicated in italics with a superscript plus sign (e.g., cdc2+); genes with a recessive

▲ FIGURE 21-11 The fission yeast S. pombe. (a) Scanning

▲ FIGURE 21-11 The fission yeast S. pombe. (a) Scanning cell cycle. Long cells are about to enter mitosis; short cells have just passed through cytokinesis, (b) Main events in the

S. pombe cell cycle. Note that the nuclear envelope does not disassemble during mitosis in S. pombe and other yeasts. [Part (a) courtesy of N. Hajibagheri.]

mutation, in italics with a superscript minus sign (e.g., cdc2~). The protein encoded by a particular gene is designated by the gene symbol in Roman type with an initial capital letter (e.g., Cdc2).

A Highly Conserved MPF-like Complex Controls Entry into Mitosis in S. pombe

Temperature-sensitive mutations in cdc2, one of several different cdc genes in pombe, produce opposite phenotypes depending on whether the mutation is recessive or dominant (Figure 21-12). Recessive mutations (cdc2~) give rise to abnormally long cells, whereas dominant mutations (cdc2D) give rise to abnormally small cells, the wee phenotype. As discussed in Chapter 9, recessive mutations generally cause a loss of the wild-type protein function; in diploid cells, both alle-les must be mutant in order for the mutant phenotype to be observed. In contrast, dominant mutations generally result in a gain in protein function, either because of overproduction or lack of regulation; in this case, the presence of only one mutant allele confers the mutant phenotype in diploid cells. The finding that a loss of Cdc2 activity (cdc2~ mutants) prevents entry into mitosis and a gain of Cdc2 activity (cdc2D mutants) brings on mitosis earlier than normal identified Cdc2 as a key regulator of entry into mitosis in ,S. pombe.

The wild-type cdc2+ gene contained in a ,S. pombe plas-mid library was identified and isolated by its ability to complement cdc2~ mutants (see Figure 21-4). Sequencing showed that cdc2+ encodes a 34-kDa protein with homology to eukaryotic protein kinases. In subsequent studies, researchers identified cDNA clones from other organisms that could complement ,S. pombe cdc2~ mutants. Remarkably, they isolated a human cDNA encoding a protein identical to sS. pombe Cdc2 in 63 percent of its residues.

▲ EXPERIMENTAL FIGURE 21-12 Recessive and dominant S. pombe cdc2 mutants have opposite phenotypes. Wild-type cell (cdc2+) is schematically depicted just before cytokinesis with two normal-size daughter cells. A recessive cdc2~ mutant cannot enter mitosis at the nonpermissive temperature and appears as an elongated cell with a single nucleus, which contains duplicated chromosomes. A dominant cdc2D mutant enters mitosis prematurely before reaching normal size in G2; thus, the two daughter cells resulting from cytokinesis are smaller than normal, that is, they have the wee phenotype.

Isolation and sequencing of another ,S. pombe cdc gene (cdc13+), which also is required for entry into mitosis, revealed that it encodes a protein with homology to sea urchin and Xenopus cyclin B. Further studies showed that a het-erodimer of Cdc13 and Cdc2 forms the ,S. pombe MPF; like Xenopus MPF, this heterodimer has protein kinase activity that will phosphorylate histone H1. Moreover, the H1 protein kinase activity rises as ,S. pombe cells enter mitosis and falls as they exit mitosis in parallel with the rise and fall in the Cdc13 level. These findings, which are completely analogous to the results obtained with Xenopus egg extracts (see Figure 21-9a), identified Cdc 13 as the mitotic cyclin in sS. pombe. Further studies showed that the isolated Cdc2 protein and its homologs in other eukaryotes have little protein kinase activity until they are bound by a cyclin. Hence, this family of protein kinases became known as cyclin-dependent kinases, or CDKs.

Researchers soon found that antibodies raised against a highly conserved region of Cdc2 recognize a polypeptide that co-purifies with MPF purified from Xenopus eggs. Thus Xenopus MPF is also composed of a mitotic cyclin (cyclin B) and a CDK (called CDK1). This convergence of findings from biochemical studies in an invertebrate (sea urchin) and a vertebrate (Xenopus) and from genetic studies in a yeast indicated that entry into mitosis is controlled by analogous mitotic cyclin-CDK complexes in all eukary-otes (see Figure 21-2, step 7). Moreover, most of the participating proteins have been found to be highly conserved during evolution.

Phosphorylation of the CDK Subunit Regulates the Kinase Activity of MPF

Analysis of additional ,S. pombe cdc mutants revealed that proteins encoded by other genes regulate the protein kinase activity of the mitotic cyclin-CDK complex (MPF) in fission yeast. For example, temperature-sensitive cdc25~ mutants are delayed in entering mitosis at the nonpermissive temperature, producing elongated cells. On the other hand, overexpression of Cdc25 from a plasmid present in multiple copies per cell decreases the length of G2 causing premature entry into mitosis and small (wee) cells (Figure 21-13a). Conversely, loss-of-function mutations in the wee1+ gene causes premature entry into mitosis resulting in small cells, whereas overproduction of Wee1 protein increases the length of G2 resulting in elongated cells. A logical interpretation of these findings is that Cdc25 protein stimulates the kinase activity of S. pombe MPF, whereas Wee1 protein inhibits MPF activity (Figure 21-13b).

In subsequent studies, the wild-type cdc25+ and wee1 + genes were isolated, sequenced, and used to produce the encoded proteins with suitable expression vectors. The deduced sequences of Cdc25 and Wee1 and biochemical studies of the proteins demonstrated that they regulate the kinase activity of S. pombe MPF by phosphorylating and dephosphorylating specific regulatory sites in Cdc2, the CDK subunit of MPF.

Deficit of Cdc25 or

Excess of Wee1

Elongated cells (increased G2)

Deficit of Wee1 or

Excess of Cdc25

Small cells (decreased G2)

S. pombe MPF

Wee1

Wee1

S. pombe MPF

Cdc25

Elongated cells (increased G2)

Small cells (decreased G2)

▲ EXPERIMENTAL FIGURE 21-13 Cdc25 and Wee1 have opposing effects on S. pombe MPF activity. (a) Cells that lack Cdc25 or Wee1 activity, as a result of recessive temperature-sensitive mutations in the corresponding genes, have the opposite phenotype. Likewise, cells with multiple copies of plasmids containing wild-type cdc25+ or wee1+, and which thus produce an excess of the encoded proteins, have opposite phenotypes. (b) These phenotypes imply that the mitotic cyclin-CDK complex is activated by Cdc25 and inhibited (—l) by Wee1. See text for further discussion.

Figure 21-14 illustrates the functions of four proteins that regulate the protein kinase activity of the S. pombe CDK. First is Cdc13, the mitotic cyclin of ,S. pombe (equivalent to cyclin B in metazoans), which associates with the CDK to form MPF with extremely low activity. Second is the Weel protein-tyrosine kinase, which phosphorylates an inhibitory tyrosine residue (Y15) in the CDK subunit. Third is another kinase, designated CDK-activating kinase (CAK), which phosphorylates an activating threonine residue (T161). When

▲ FIGURE 21-14 Regulation of the kinase activity of S. pombe mitosis-promoting factor (MPF). Interaction of mitotic cyclin (Cdc13) with cyclin-dependent kinase (Cdc2) forms MPF The CDK subunit can be phosphorylated at two regulatory sites: by Wee1 at tyrosine-15 (Y15) and by CDK-activating kinase (CAK) at threonine-161 (T161). Removal of the phosphate on Y15

both residues are phosphorylated, MPF is inactive. Finally, the Cdc25 phosphatase removes the phosphate from Y15, yielding highly active MPF. Site-specific mutagenesis that changed the Y15 in ,S. pombe CDK to a phenylalanine, which cannot be phosphorylated, produced mutants with the wee phenotype, similar to that of wee1~ mutants. Both mutations prevent the inhibitory phosphorylation at Y15, resulting in the inability to properly regulate MPF activity, and, consequently, premature entry into mitosis.

Conformational Changes Induced by Cyclin Binding and Phosphorylation Increase MPF Activity

Unlike both fission and budding yeasts, each of which produce just one CDK, vertebrates produce several CDKs (see Table 21-1). The three-dimensional structure of one human cyclin-dependent kinase (CDK2) has been determined and provides insight into how cyclin binding and phosphoryla-tion of CDKs regulate their protein kinase activity. Although the three-dimensional structures of the S. pombe CDK and most other cyclin-dependent kinases have not been determined, their extensive sequence homology with human CDK2 suggests that all these CDKs have a similar structure and are regulated by a similar mechanism.

Unphosphorylated, inactive CDK2 contains a flexible region, called the T-loop, that blocks access of protein substrates to the active site where ATP is bound (Figure 21-15a). Steric blocking by the T-loop largely explains why free CDK2, unbound to cyclin, has no protein kinase activity. Unphospho-rylated CDK2 bound to one of its cyclin partners, cyclin A, has minimal but detectable protein kinase activity in vitro, although it may be essentially inactive in vivo. Extensive interactions between cyclin A and the T-loop cause a dramatic shift in the position of the T-loop, thereby exposing the CDK2 active site (Figure 21-15b). Binding of cyclin A also shifts the position of the a1 helix in CDK2, modifying its substrate-binding surface. Phosphorylation of the activating threonine, located by Cdc25 phosphatase yields active MPF in which the CDK subunit is monophosphorylated at T161. The mitotic cyclin subunit contributes to the specificity of substrate binding by MPF, probably by forming part of the substrate-binding surface (crosshatch), which also includes the inhibitory Y15 residue.

Figure 21-14 illustrates the functions of four proteins that regulate the protein kinase activity of the S. pombe CDK. First is Cdc13, the mitotic cyclin of ,S. pombe (equivalent to cyclin B in metazoans), which associates with the CDK to form MPF with extremely low activity. Second is the Weel protein-tyrosine kinase, which phosphorylates an inhibitory tyrosine residue (Y15) in the CDK subunit. Third is another kinase, designated CDK-activating kinase (CAK), which phosphorylates an activating threonine residue (T161). When

(b) Low-activity cyclin A-CDK2

a1 Helix

T loop a1 Helix

T loop

(b) Low-activity cyclin A-CDK2

(c) High-activity cyclin A-CDK2

▲ FIGURE 21-15 Structural models of human CDK2, which is homologous to the S. pombe cyclin-dependent kinase (CDK). (a) Free, inactive CDK2 unbound to cyclin A. In free CDK2, the T-loop blocks access of protein substrates to the •y-phosphate of the bound ATP shown as a ball-and-stick model. The conformations of the regions highlighted in yellow are altered when CDK is bound to cyclin A. (b) Unphosphorylated, low-activity cyclin A-CDK2 complex. Conformational changes induced by binding of a domain of cyclin A (green) cause the T-loop to pull away from the active site of CDK2, so that substrate proteins can bind. The a1 helix in CDK2, which interacts extensively with cyclin A, moves several angstroms into the catalytic cleft, repositioning key catalytic side chains required for the phosphorotransfer reaction to substrate specificity. The red ball marks the position equivalent to threonine 161 in S. pombe Cdc2. (c) Phosphorylated, high-activity cyclin A-CDK2 complex. The conformational changes induced by phosphorylation of the activating threonine (red ball) alter the shape of the substrate-binding surface, greatly increasing the affinity for protein substrates. [Courtesy of P D. Jeffrey. See A. A. Russo et al., 1996, Nature Struct. Biol. 3:696.]

in the T-loop, causes additional conformational changes in the cyclin A-CDK2 complex that greatly increase its affinity for protein substrates (Figure 21-15c). As a result, the kinase activity of the phosphorylated complex is a hundredfold greater than that of the unphosphorylated complex.

The inhibitory tyrosine residue (Y15) in the ,S. pombe CDK is in the region of the protein that binds the ATP phosphates. Vertebrate CDK2 proteins contain a second inhibitory residue, threonine-14 (T14), that is located in the same region of the protein. Phosphorylation of Y15 and T14 in these proteins prevents binding of ATP because of electrostatic repulsion between the phosphates linked to the protein and the phosphates of ATP. Thus these phosphorylations inhibit protein kinase activity even when the CDK protein is bound by a cyclin and the activating residue is phosphorylated.

Other Mechanisms Also Control Entry into Mitosis by Regulating MPF Activity

So far we have discussed two mechanisms for controlling entry into mitosis: (a) regulation of the concentration of mitotic cyclins as outlined in Figure 21-10 and (b) regulation of the kinase activity of MPF as outlined in Figure 21-14. Further studies of sS. pombe mutants with altered cell cycles have revealed additional genes whose encoded proteins directly or indirectly influence MPF activity. At present it is clear that MPF activity in S. pombe is regulated in a complex fashion in order to control precisely the timing of mitosis and therefore the size of daughter cells.

Enzymes with activities equivalent to ,S. pombe Wee1 and Cdc25 have been found in cycling Xenopus egg extracts. The

Xenopus Wee1 tyrosine kinase activity is high and Cdc25 phosphatase activity is low during interphase. As a result, MPF assembled from Xenopus CDK1 and newly synthesized mitotic cyclin is inactive. As the extract initiates the events of mitosis, Wee1 activity diminishes and Cdc25 activity increases so that MPF is converted into its active form. Although cyclin B is the only protein whose synthesis is required for the cycling of early Xenopus embryos, the activities of other proteins, including Xenopus Wee1 and Cdc25, must be properly regulated for cycling to occur. In its active form, Cdc25 is phosphorylated. Its activity is also controlled by additional protein kinases and phosphatases.

MPF activity also can be regulated by controlling transcription of the genes encoding the proteins that regulate MPF activity. For example, after the initial rapid synchronous cell divisions of the early Drosophila embryo, all the mRNAs are degraded, and the cells become arrested in G2. This arrest occurs because the Drosophila homolog of Cdc25, called String, is unstable. The resulting decrease in String phosphatase activity maintains MPF in its inhibited state, preventing entry into mitosis. The subsequent regulated entry into mitosis by specific groups of cells is then triggered by the regulated transcription of the string gene.

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