Sensory Transduction in Vision, Olfaction, and Gustation:- Levels of Cyclin-Dependent Protein Kinases Oscillate
The timing of the cell cycle is controlled by a family of protein kinases with activities that change in response to cellular signals. By phosphorylating specific proteins at precisely timed intervals, these protein kinases or chestrate the metabolic activities of the cell to produce orderly cell division. The kinases are heterodimers with a regulatory subunit, cyclin, and a catalytic subunit, cyclin-dependent protein kinase (CDK). In the absence of cyclin, the catalytic subunit is virtually inactive. When cyclin binds, the catalytic site opens up, a residue essential to catalysis becomes accessible (Fig. 12–42), and the activity of the catalytic subunit increases 10,000-fold. Animal cells have at least ten different cyclins (designated A, B, and so forth) and at least eight cyclin-dependent kinases (CDK1 through CDK8), which act in various combinations at specific points in the cell cycle. Plants also use a family of CDKs to regulate their cell division.
FIGURE 12–42 Activation of cyclin-dependent protein kinases (CDKs) by cyclin and phosphorylation. CDKs, a family of related enzymes, are active only when associated with cyclins, another protein family. The crystal structure of CDK2 with and without cyclin reveals the basis for this activation. (a) Without cyclin (PDB ID 1HCK), CDK2 folds so that one segment, the T loop (red), obstructs the binding site for protein substrates and thus inhibits protein kinase activity. The binding site for ATP (blue) is also near the T loop. (b) When cyclin binds (PDB ID 1FIN), it forces conformational changes that move the T loop away from the active site and reorient an amino-terminal helix (green), bringing a residue critical to catalysis (Glu51) into the active site. (c) Phosphorylation of a Thr residue (dark orange space-filling structure) in the T loop produces a negatively charged residue that is stabilized by interaction with three Arg residues (red ball-and-stick structures), holding CDK in its active conformation (PDB ID 1JST).
In a population of animal cells undergoing synchro nous division, some CDK activities show striking oscillations (Fig. 12–43). These oscillations are the result of four mechanisms for regulating CDK activity: phosphorylation or dephosphorylation of the CDK, controlled degradation of the cyclin subunit, periodic synthesis of CDKs and cyclins, and the action of specific CDK inhibiting proteins.
Regulation of CDKs by Phosphorylation The activity of a CDK is strikingly affected by phosphorylation and de phosphorylation of two critical residues in the protein (Fig. 12–44a). Phosphorylation of Tyr15 near the amino terminus renders CDK2 inactive; the P–Tyr residue is in the ATP-binding site of the kinase, and the negatively charged phosphate group blocks the entry of ATP. A specific phosphatase dephosphorylates this P–Tyr residue, permitting the binding of ATP. Phosphorylation of Thr160 in the “T loop” of CDK, catalyzed by the CDK activating kinase, forces the T loop out of the substrate binding cleft, permitting substrate binding and catalytic activity.
FIGURE 12–43 Variations in the activities of specific CDKs during the cell cycle in animals. Cyclin E–CDK2 activity peaks near the G1 phase–S phase boundary, when the active enzyme triggers synthesis of enzymes required for DNA synthesis (see Fig. 12–46). Cyclin A–CDK2 activity rises during the S and G2 phases, then drops sharply in the M phase, as cyclin B–CDK1 peaks.
FIGURE 12–44 Regulation of CDK by phosphorylation and proteolysis. (a) The cyclin-dependent protein kinase activated at the time of mitosis (the M phase CDK) has a “T loop” that can fold into the substrate-binding site. When Thr160 in the T loop is phosphorylated, the loop moves out of the substrate-binding site, activating the CDK manyfold. (b) The active cyclin-CDK complex triggers its own inactivation by phosphorylation of DBRP (destruction box recognizing protein). DBRP and ubiquitin ligase then attach several molecules of ubiquitin (U) to cyclin, targeting it for destruction by proteasomes, proteolytic enzyme complexes.
One circumstance that triggers this control mechanism is the presence of single-strand breaks in DNA, which leads to arrest of the cell cycle in G2. A specific protein kinase (called Rad3 in yeast), which is activated by single-strand breaks, triggers a cascade leading to the inactivation of the phosphatase that dephosphorylates Tyr15 of CDK. The CDK remains inactive and the cell is arrested in G2. The cell will not divide until the DNA is repaired and the effects of the cascade are reversed.
Controlled Degradation of Cyclin Highly specific and precisely timed proteolytic breakdown of mitotic cyclins regulates CDK activity throughout the cell cycle. Progress through mitosis requires first the activation then the destruction of cyclins A and B, which activate the catalytic subunit of the M-phase CDK. These cyclins contain near their amino terminus the sequence Arg–Thr–Ala–Leu–Gly–Asp–Ile–Gly–Asn, the “destruction box,” which targets them for degradation. (This us age of “box” derives from the common practice, in diagramming the sequence of a nucleic acid or protein, of enclosing within a box a short sequence of nucleotide or amino acid residues with some specific function. It does not imply any three-dimensional structure.) The protein DBRP (destruction box recognizing protein) recognizes this sequence and initiates the process of cy clin degradation by bringing together the cyclin and another protein, ubiquitin. Cyclin and activated ubiquitin are covalently joined by the enzyme ubiquitin ligase (Fig. 12–44b). Several more ubiquitin molecules are then appended, providing the signal for a proteolytic en zyme complex, or proteasome, to degrade cyclin. What controls the timing of cyclin breakdown? A feedback loop occurs in the overall process shown in Figure 12–44. Increased CDK activity activates cyclin proteolysis. Newly synthesized cyclin associates with and activates CDK, which phosphorylates and activates DBRP. Active DBRP then causes proteolysis of cyclin. Lowered [cyclin] causes a decline in CDK activity, and the activity of DBRP also drops through slow, constant dephosphorylation and inactivation by a DBRP phosphatase. The cyclin level is ultimately restored by syn thesis of new cyclin molecules. The role of ubiquitin and proteasomes is not limited to the regulation of cyclin; as we shall see in Chapter 27, both also take part in the turnover of cellular proteins, a process fundamental to cellular housekeeping. Regulated Synthesis of CDKs and Cyclins The third mechanism for changing CDK activity is regulation of the rate of synthesis of cyclin or CDK or both. For example, cy clin D, cyclin E, CDK2, and CDK4 are synthesized only when a specific transcription factor, E2F, is present in the nucleus to activate transcription of their genes. Syn thesis of E2F is in turn regulated by extracellular signals such as growth factors and cytokines (inducers of cell division), compounds found to be essential for the division of mammalian cells in culture. These growth factors induce the synthesis of specific nuclear transcription factors essential to the production of the enzymes of DNA synthesis. Growth factors trigger phos phorylation of the nuclear proteins Jun and Fos, transcription factors that promote the synthesis of a variety of gene products, including cyclins, CDKs, and E2F. In turn, E2F controls production of several enzymes essential for the synthesis of deoxynucleotides and DNA, enabling cells to enter the S phase (Fig. 12–45).
Inhibition of CDKs Finally, specific protein inhibitors bind to and inactivate specific CDKs. One such protein is p21, which we discuss below.
FIGURE 12–45 Regulation of cell division by growth factors. The path from growth factors to cell division leads through the enzyme cascade that activates MAPK; phosphorylation of the nuclear transcription factors Jun and Fos; and the activity of the transcription fac tor E2F, which promotes synthesis of several enzymes essential for DNA synthesis.
These four control mechanisms modulate the activity of specific CDKs that, in turn, control whether a cell will divide, differentiate, become permanently quiescent, or begin a new cycle of division after a period of quiescence. The details of cell cycle regulation, such as the number of different cyclins and kinases and the combinations in which they act, differ from species to species, but the basic mechanism has been conserved in the evolution of all eukaryotic cells.
