Akt (also known as PKB or RAC-PK) is an intracellular serine/threonine kinase involved in regulating cell survival. Although this makes it a promising target for the discovery of drugs to treat human cancer, a complicating factor may be the role played by Akt in insulin signalling. Two human isoforms, 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-g or PKB-g). We describe the identification of the corresponding human isoform of Akt-3. The gene encoding human Akt-3 was localized to chromosome 1q43±44. The predicted protein sequence is 83% identical to human Akt-1 and 78% 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 phosphorylation site (Ser472) thought to be involved in the activation of Akt kinases. In addition to phosphorylation of Ser472, phosphorylation of Thr305 also appears to contribute to the activation of Akt-3 because mutation of both these residues to aspartate increased the catalytic activity of Akt-3, whereas mutation to alanine inhibited activation. Akt-3 activity could be inhibited by the broad spectrum kinase inhibitor staurosporine and by the PKC inhibitor 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 muscle, suggesting that its principle function may not be in regulating insulin signalling. These observations suggest that Akt-3 is a promising target for the discovery of novel chemotherapeutic agents which do not interfere with insulin signalling.
Raf-1, the cellular homolog of the v-raf oncoprotein, is a ubiquitously expressed serine/threonine kinase which serves as a central interface in the transmission of mitogenic signals from the cell membrane to the nucleus. Raf-1 is activated by various growth factors, and its function has been shown to be required for transformation by several classes of oncogenes, including ligands, tyrosine kinase receptors, Ras proteins, and src family tyrosine kinases (recently reviewed in references 18, 50, and 98). Recent investigations have charted a pathway indicating how Raf-1 links membrane-bound signalling molecules to nuclear transcription factors. Upon activation, many growth factor receptors associate with adaptor proteins, such as grb-2, crk, and shc, which in turn recruit guanine nucleotidereleasing proteins (GNRP), such as SOS and C3G, to the plasma membrane. GNRP activate Ras proteins by mediating the exchange of GDP to GTP (29, 62; for reviews, see references 63 and 68). GTP-loaded Ras proteins can bind to the N-terminal region of Raf-1 with high affinity (reviewed in reference 68), causing the translocation of Raf-1 from the cytosol to the membrane, where Raf-1 is exposed to activators (58, 89). The nature of the physiological Raf-1 activator (s) is not yet clear but might include protein kinase C (6, 52, 87) and src family tyrosine kinases (26,60,94,99). Activated Raf-1 in turn can phosphorylate Mek, a dual-specificity kinase which activates the extracellular signal-regulated kinases Erk-1 and -2 by phosphorylation on tyrosine and threonine residues (for reviews, e.g., see references 18, 61, and 98). Activated Erks can translocate to the nucleus and phosphorylate transcription factors. The best-studied nuclear target of Erks is the ternary complex factor, which is required for induction of the c-fos gene (34, 54). The c-Fos protein associates with c-Jun to form the AP-1 transcription factor (reviewed in reference 16), which is a main mediator of v-raf transformation (53,54,73). The activation of this signalling cascade leading from the cell membrane to the nucleus seems to be both necessary and sufficient for transformation of NIH 3T3 fibroblasts (14).A number of recent reports have identified the cyclic AMP (cAMP)-dependent kinase protein kinase A (PKA) as a negative regulator of this pathway. Activation of PKA blocks the activation of Erks in several different cell types, including fibroblasts and smooth muscle cells (4,11,36,38,40,80,91,101). PKA does not affect the activity of Ras, Mek, or mitogenactivated protein kinase (MAPK) (11, 101) but was shown to both inhibit Ras-dependent activation of Raf-1 (4, 11, 101) and downregulate Raf-1 kinase activity directly (38). Phosphorylation of the Raf-1 regulatory domain by PKA decreases the affinity of Raf-1 for activated GTP-loaded Ras, thereby preventing the activation of 101). In addition, phosphorylation of the kinase domain directly suppresses the catalytic activity of the Raf-1 kinase domain (38). This type of inhibition is dominant, as both activated Raf-1...
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