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We have cloned human protein kinase B␥ (PKB␥) and found that it contains two regulatory phosphorylation sites, Thr 305 and Ser 472 , which correspond to Thr 308 and Ser 473 of PKB␣. Thus it differs significantly from the previously published rat PKB␥. We have also isolated a similar clone from a mouse cDNA library. In human tissues, PKB␥ is widely expressed as two transcripts. A mutational analysis of the two regulatory sites of human PKB␥ showed that phosphorylation of both sites, occurring in a phosphoinositide 3-kinase-dependent manner, is required for full activity. Our results suggest that the two phosphorylation sites act in concert to produce full activation of PKB␥, similar to PKB␣. This contrasts with rat PKB␥, which is thought to be regulated by 3-phosphoinositide-dependent protein kinase 1 alone. Three members of the protein kinase B (PKB)1 subfamily of second-messenger regulated serine/threonine protein kinases have been identified and termed ␣, ␤, and ␥ (1-4). The isoforms are homologous, particularly in regions encoding the N-terminal pleckstrin homology (PH) and the catalytic domains. PKBs are activated by phosphorylation events occurring in response to phosphoinositide 3-kinase (PI3K) signaling (5-8). PI3K phosphorylates membrane inositol phospholipids, generating the second messengers phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (reviewed in Ref. 9), which have been shown to bind to the PH domain of PKB (10, 11). The current model of PKB activation proposes recruitment of the enzyme to the membrane by 3Ј-phosphorylated phosphoinositides, where phosphorylation of the regulatory sites of PKB by the upstream kinases occurs (12)(13)(14).Phosphorylation of PKB␣/␤ occurs on two regulatory sites, Thr 308/309 in the activation loop in the catalytic domain and Ser 473/474 in the C-terminal domain (15, 16). The upstream kinase, which phosphorylates PKB␣ at the activation loop site Thr 308 , has been cloned and termed 3-phosphoinositidedependent protein kinase 1 (PDK1; . PDK1 phosphorylates not only PKB␣, but also equivalent sites in the p70 ribosomal S6 kinase (21,22), protein kinase A (23), and protein kinase C (24). The upstream kinase phosphorylating the second regulatory site of PKB␣/␤, Ser 473/474 , has not been identified yet, but a recent report implies a role for the integrin-linked kinase (ILK-1), a serine/threonine protein kinase (25).Only a few studies have been reported on the third member of the PKB family, PKB␥, and these all involved a clone originating from a rat brain cDNA library (4, 26). A major feature distinguishing rat PKB␥ from the otherwise very similar ␣ and ␤ isoforms is the C terminus, which is truncated by 23 amino acids and lacks Ser 473/474 , one of the two phosphorylation sites essential for activation of PKB␣ and ␤ (15,16). Consequently, it has been suggested that rat PKB␥ activation depends solely on the upstream kinase PDK1. We now report the cloning and characterization of human PKB␥. This isoform differs significantly from the rat ...

In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyltransferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNATI mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes.Amine acetylation in insects by the enzyme arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) is involved in at least three different physiological functions: (i) It appears to be the major route of neurotransmitter catabolism (1-4), inactivating biogenic amines including tryptamine, tyramine, octopamine, serotonin, and norepinephrine. (ii) An acetylated amine, acetyldopamine, is subsequently oxidized to the respective quinone, which cross-links different proteins and/or chitin, resulting in stabilization and hardening (sclerotization) of the insect cuticle (5). (iii) Acetylation of serotonin plays an important role in the regulation of photoperiodically influenced physiological and behavioral processes (6). The major regulatory hormone is melatonin (7, 8), which is synthesized by the methylation of acetylserotonin. In vertebrates, melatonin is secreted periodically with high concentration at night, caused by a 10-to 100-fold nocturnal increase in aaNAT activity (8). The final steps of aaNAT activation are still unclear and presumably involve new RNA and protein synthesis (9-12). All living organisms are influenced by environmental factors such as light and dark and thus need mechanisms to coordinate their physiological processes in response to seasonal changes. Serotonin, aaNAT activity, and melatonin have indeed been found in several organs (eye, optic lobe, and brain) of various invertebrate species in which melatonin concentration also shows a rhythmic behavior (13). However, it is not known if aaNAT activity and melatonin are subject to diurnal variation in Drosophila melanogaster. aaNAT activity has been described in several insect species (14-18), including D. melanogaster (2, 19), but none of these enzymes could be purified to derive antibodies or sufficient internal sequence information for cloning. We have purified an aaNAT from D. melanogaster using tryptam...

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