The second messenger cAMP, synthesized by adenylyl cyclase, transduces a wide variety of physiological signals in different cell types in multicellular animals. Virtually all of the diverse effects of cAMP are mediated through activation of protein kinase A (PKA), also called cAMP-dependent protein kinase, which phosphorylates multiple intracellular target proteins expressed in different cell types. Inactive PKA is a tetramer consisting of two regulatory (R) subunits and two catalytic (C) subunits (Figure 15-27a). Each R subunit contains a pseudosubstrate domain whose sequence resembles that of a peptide substrate and binds to the active site in the catalytic domain but is not phosphorylated; thus the pseudosubstrate domain inhibits the activity of the catalytic subunits. Inactive PKA is turned on by binding of cAMP. Each R subunit has two distinct cAMP-binding sites, called CNB-A and CNB-B (Figure 15-27b). Binding of cAMP to both sites causes a conformational change in the R subunit, including its pseudosubstrate domain, so that it can no longer bind to and inhibit the catalytic domain, and thus releases it, instantly activating its kinase activity (Figure 15-27c).
FIGURE 15-27 Structure of PKA and its activation by cAMP. (a) PKA consists of two regulatory (R) subunits and two catalytic (C) subunits. When cAMP (red triangle) binds to the regulatory subunit, the catalytic subunit is released, thus activating PKA. (b) The two regulatory subunits form a dimer, joined by a dimerization/docking domain and a flexible linker to which an A kinase–associated protein (AKAP; see Figure 15-31) can bind. Each R subunit has two cAMP-binding domains, CNB-A and CNB-B, and a binding site for a catalytic subunit (arrow). (c) Binding of cAMP to the CNB-A domain causes a subtle conformational change that displaces the C subunit from the R subunit, leading to its activation. Without bound cAMP, one loop of the CNB-A domain (purple) is in a conformation that can bind the catalytic (C) subunit. A glutamate (E200) and arginine (R209) residue participate in binding of cAMP (red), which causes a conformational change (green) in the loop that prevents binding of the loop to the C subunit.
[Part (b) cAMP, CNB-A, and CNB-B data, and part (c) cAMP bound data from Y. Su et al., 1995, Science 269:807, PDB ID 1gs. Part (b) docking domain data from P. Banky et al., 2003, J. Mol. Biol. 330:1117, PDB ID 2ezw. Part (c) catalytic subunit bound data from C. Kim, N. H. Xuong, and S. S. Taylor, 2005, Science 307:690, PDB ID 1u7e.]
Binding of cAMP by an R subunit of PKA occurs in a cooperative fashion; that is, binding of the first cAMP molecule to CNB-B lowers the Kd for binding of the second cAMP to CNB-A. Thus small changes in the level of cytosolic cAMP can cause proportionately large changes in the number of dissociated C subunits and, hence, in cellular kinase activity. Rapid activation of enzymes by signal-triggered dissociation of an inhibitor is a common feature of many signaling pathways.