Fig. 2. (A) Regulation of GSK-3ß activity by serine phosphorylation. In the resting cell, GSK-3ß is constitutively active. Both unprimed substrates and substrates phosphorylated by a priming kinase (PK) are capable of being phosphorylated by the active GSK-3ß. The priming phospho-residue at position N + 4, binds a pocket of positive charge arising from the arginine (R) and lysine (K) residues indicated. This directs a serine or threonine at position N to the active catalytic site (C.S.). When an inactivating kinase (IK) such as PKB/Akt phosphorylates GSK-3ß on serine 9 (S9), the phosphorylated N-terminus becomes a primed pseudo-substrate that occupies the positive binding pocket and active site of the enzyme, acting as a competitive inhibitor for true substrates. This prevents phosphorylation of any substrates. (B) Effect of mutating arginine 96 to alanine (R96A) on GSK-3ß activity. Since arginine 96 is a crucial component of the positive pocket that binds primed substrates, its mutation to an uncharged alanine residue disrupts the pocket so that primed substrates can no longer bind. The enzyme retains activity. Also, the S9-phosphorylated pseudosubstrate is no longer capable of inactivating the enzyme. As a consequence, GSK-3ß, whether S9-phosphorylated or not, can phosphorylate unprimed substrates, but not primed substrates. Note that unprimed and primed substrates interact with GSK-3 through different interfaces.