Molecular cloning, expression and characterization of the human serine/threonine kinase Akt-3

Akt (also known as PKB or RAC-PK) is an intracellular serine/threonine kina se involved in regulating cell survival. Although this makes it a promising target for,the discovery of drugs to treat human cancer, a complicating fa ctor may be the role played by Akt in insulin, signalling. Two human isofor ms, Akt-1 and Akt-2, have been described previously and a third isoform has been-identified in rats (here termed Akt-3, but also called RAC-PK-gamma o r PKB-gamma). We describe the identification of the corresponding human iso form of Akt-3. The gene encoding human Akt-3 was localized to chromosome 1q 43-44. The predicted protein sequence is 83% identical to human Akt-1 and 7 8% identical to human Akt-2, and contains a pleckstrin homology domain and a kinase domain. In contrast to the published rat Akt-3 isoform, human and mouse Akt-3 also possess a C-terminal 'tail' that contains a phosphorylatio n site (Ser472) thought to be involved in the activation of Akt kinases. In addition to phosphorylation of Ser472, phosphorylation of Thr305 also appe ars to contribute to the activation of Akt-3 because mutation of both these residues to aspartate increased the catalytic activity of Akt-3, whereas m utation to alanine inhibited activation. Akt-3 activity could be inhibited by:the broad spectrum kinase inhibitor staurosporine and by the PKC inhibit or Ro 31-8220, but not by other PKC or PKA inhibitors tested. Although Akt- 3 is expressed widely, it is not highly expressed in liver or skeletal musc le, suggesting that its principle function may not be in regulating insulin signalling. These observations suggest that Akt-3 is a promising target fo r the discovery of novel chemotherapeutic agents which db not interfere wit h insulin signalling.