BIOLOGICAL OVERVIEW

Organism size is determined by a tightly regulated mechanism that coordinates cell growth, cell proliferation and cell death. The Drosophila insulin receptor/Chico/Dp110 pathway regulates cell and organism size. Chico, an adaptor protein that binds to the Insulin-like receptor, and Phosphotidylinositol 3 kinase 92E (Dp110), an enzyme that phosphorylates lipids, are both involved in transmitting insulin receptor signals downstream to cellular effectors. The subject of this overview, the phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt1 (also known as protein kinase B or PKB) affects cell and organ size in Drosophila in a cell autonomous manner (Verdu, 1999). PKB has a PH domain that binds 3-phosphorylated inositol lipids (phosphatidylinositol 3,4,5-trisphosphate also known as PIP3), and the translocation of the mammalian homolog of Drosophila Akt1 to the plasma membrane is an important part of its activation. PKB is also phosphorylated by a PIP3-activated phosphoinositide-dependent protein kinase (PDK-1), which has a PH domain that binds PIP3. Thus there are two independent contributions of 3-phosphorylated inositol lipids to the activation of PKB, one via PDK-1 and the other involving PKB itself (Irvine, 1998 and references therein). Akt appears to stimulate intracellular pathways that specifically regulate cell and compartment size independent of cell proliferation in vivo (Verdu, 1999).

To determine whether Drosophila Akt1 participates in insulin-receptor signal transduction, Akt1 activity was measured in Schneider (S2) cells. Insulin stimulates Akt1 activity sevenfold in S2 cells overexpressing a wild-type Akt1 transgene. Furthermore, membrane localization of Akt1 by addition of an src myristoylation sequence to its amino terminus is sufficient to confer constitutive kinase activity. In contrast, kinase-deficient Akt1 shows activity neither in the basal state nor after insulin stimulation, thus indicating that the measured phosphotransferase activity is not due to a contaminating kinase. These observations confirm that Akt1 is regulated in a way similar to that of its mammalian homolog. Consistent with this proposal, pretreatment with the PI(3)K inhibitor wortmannin blocks Akt1 activation by insulin. These data indicate that, as in mammalian cells, Drosophila PI(3)K is a component required for mediating the activation of Akt1 (Verdu, 1999).

To determine whether ectopic expression of Akt1 increases the size of tissues, Akt1 was targeted to the wing using a 71BGAL4 line. This resulted in a marked enlargement of the wing imaginal disc and an expansion of the surface of the adult wing blade as well as an increase in vein thickness. This increase in size is often accompanied by a mild disruption of the proximo-distal alignment characteristic of the hairs present on the wing-blade surface. Morphometric analysis of 71BGAL4/UAS-Akt1 wings reveals a 29% increase in wing surface area. Furthermore, ectopic expression of Akt1 along the anteroposterior boundary of the wing imaginal disc results in enlargement of only the corresponding region of the adult wing. In spite of the increased size of the wing in 71BGAL4/UAS-Akt1 flies, there is no change in the number of cells, resulting in a cell density in 71BGAL4/UAS-Akt1 flies that is 15% lower than that in 71BGAL4/+ controls. Together, these observations show that ectopic expression of Akt1 increases the size of the wing imaginal disc, leading to enlargement of the adult wing. The question of whether the effect of Akt1 on compartment growth in the wing is cell autonomous was addressed further. Targeting of Akt1 to the posterior compartment of the wing imaginal disc with an engrailed-GAL4 line results in a marked expansion of this region, whereas the anterior compartment remains unaffected (Verdu, 1999).

To evaluate the Akt1-selective increase in cell size more quantitatively, Akt1 was expressed in the posterior compartment of wing imaginal discs; measured were compartment areas, cell size and cell number, the latter two by flow cytometry. Expression or inactivation of cell-cycle regulators, such as E2F, RBF and Cdc2, in the posterior compartment affects cell size and number without altering compartment size. Akt1 expression increases the area occupied by the posterior compartment concomitant with a marked enlargement of its cells as measured by forward light scatter. Strikingly, no changes in the number of cells in the posterior compartment are detected. Thus, overexpression of Akt1 affects compartment size by altering cell growth without a concomitant increase in the final number of cells within the compartment. Studies of mammalian cells have indicated that Akt may positively regulate cell-cycle progression. However, in the wing imaginal disc, no differences were found in cell proliferation between control cells in the anterior compartment and cells expressing Akt1 in the posterior compartment, as judged by the pattern or frequency of bromodeoxyuridine incorporation (Verdu, 1999).

Akt overrides G1 arrest induced by PTEN (see Drosophila Pten) and by interleukin-2 deprivation in cell-culture models. To determine whether ectopic Akt1 could bypass cell-cycle arrest in imaginal tissues, a population of physiologically arrested cells in the wing imaginal disc, the zone of non-proliferating cells (ZNC), was studied. Expression of positive regulators of the cell cycle, such as the phosphatase Cdc25^string and cyclin E, bypasses both G1 and G2 arrests in the ZNC. Interestingly, Akt1 expression in the posterior compartment does not rescue the cells of the ZNC from their G1 arrest. As a more quantitative assay of Akt1 effects on cell-cycle progression, wing imaginal discs ubiquitously expressing Akt1 were dissected and cellular DNA content was measured by flow cytometry. The proportions of cells in G1, S and G2 phase remain indistinguishable in cells expressing Akt1 and wild-type cells, despite the differences in compartment size (Verdu, 1999).

Compartments function as an independent units of growth and size control. Ectopic expression of Akt1 overrides the intrinsic control mechanisms regulating the final size of posterior compartment. To circumvent potential compartment controls on cell number, clones of cells overexpressing Akt1 were generated in the wing imaginal disc. Clone size was assessed 48 h after induction by heat-shock. Akt1 markedly increases clonal size through an enlargement of the cells rather than an increase in the cell number. As a more sensitive assay of cell number, clones of cells expressing Akt1 were induced in the wing disc 72 h after egg deposition; cell number was assessed 48 h later. Analysis reveals that the increase in clonal size induced by ectopic Akt1 expression is due to a selective increase in cell size but not cell number. Thus, it is concluded that Akt1 affects compartment size by increasing cell growth (that is, cell size) without altering cell proliferation (Verdu, 1999).

Several lines of evidence indicate a requirement for components of the protein-synthetic regulatory apparatus for cell growth. The large-cell and small-cell phenotypes resulting from increasing or removing Akt activity, respectively, are consistent with concomitant alterations in the translational machinery. In mammals, Akt appears to influence the rate of protein synthesis through mTOR (for mammalian target of rapamycin)-mediated activation of p70S6kinase (see Drosophila RPS6-p70-protein kinase) and inhibition of the 4E-binding protein-1 (4E-BP1 or PHAS-1), a repressor of translation initiation. These results implicate Akt as an activator of messenger RNA translation and indicate that regulation of this pathway could be relevant to the ability of Akt to promote cell growth in vivo. A critical question is whether increases in protein synthesis are merely permissive for expansion of cell size, implying the existence of a distinct growth-regulatory mechanism, or whether Akt-dependent enhancement of protein translation is in itself sufficient to cause an increase in organ size. Alternatively, the augmentation in cell growth produced by Akt could be the result of activation of a concerted anabolic program, for which protein synthesis would be a vital component (Verdu, 1999 and references therein).

An important question arising from this and other papers is how signaling from the insulin receptor regulates compartment size. From the data presented here it can be concluded that manipulation of Akt levels affects compartment size by increasing cell growth without significant changes in cell number. Similar findings have been obtained from study of wing discs with reduced levels of S6 kinase (Montagne, 1999). The insulin receptor, Chico and Dp110 appear to influence both cell size and number in the Drosophila wing. Thus, a plausible scenario is that the pathway bifurcates directly upstream of Akt, which is required for cell growth (through a Drosophila TOR, S6 kinase and 4E-BP1), while a second branch mediates cell proliferation through a parallel pathway. However, it is not yet clear that activation of the insulin-receptor signaling pathway promotes cell proliferation in Drosophila. Reduction in levels of 1) the insulin receptor, 2) Chico or 3) Dp110 negatively affects cell growth and cell number. Nonetheless, it remains unclear whether this is a direct result of modulation of the cell-cycle machinery, or secondary to an impairment in cell growth. Inadequate cell growth may well function as a mitotic checkpoint, or render the cell more susceptible to apoptosis as cell division proceeds unabated. Either mechanism would result in a decrease in cell number. Interestingly, ectopic expression of Dp110 in clones of cells in the wing imaginal disc results in a dramatic increase in cell and clone size, with no effects in cell number. In any case, clearly the phenotypes resulting from ectopic expression of cell-cycle regulators in the wing disc do not resemble those reported for ectopic expression of Dp110, Akt and S6 kinase. Thus, the effects of the insulin-receptor pathway on cell growth are unlikely to be secondary to alterations in cell cycle, but probably represent the major biological output for Chico, Dp110 and Akt in Drosophila. Other regulatory pathways probably function as primary determinants of proliferation (Verdu, 1999 and references therein).

PDK1 regulates growth through Akt and S6K in Drosophila

The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in C. elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. In Drosophila, dPDK1 (FlyBase name: Protein kinase 61C) controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K (FlyBase name: RPS6-p70-protein kinase). Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN (FlyBase name: Protein kinase related to protein kinase N), encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K (Rintelen, 2001).

To analyze the function of dPDK1 in Drosophila, gain- and loss-of-function alleles of the kinase were generated. Drosophila contains a single gene that encodes a kinase that is highly homologous to PDK1 in its primary sequence and its domain structure. Initially, two EP transposable elements in the 5' region of the endogenous Drosophila PDK1 gene dPDK1 were identified. These EP elements drive expression of dPDK1 under the control of the Gal4 system, allowing a test whether dPDK1 and dAkt cooperate in promoting growth in Drosophila. Overexpression of either kinase in the eye imaginal disc during the last cell division cycle and subsequent differentiation shows little effect on the size or the structure of the eye. Co-overexpression of dAkt and dPDK1, however, leads to a significant increase in eye size. Furthermore, analysis of clones of cells in the eye overexpressing dPDK1 and/or dAkt reveals that the observed effect on cell size is strictly autonomous. These results indicate that overexpression of dPDK1 does not interfere with the normal differentiation of eye disc cells and that it promotes local growth through dAkt activation (Rintelen, 2001).

To generate loss-of-function alleles of dPDK1, the dominant eye size phenotype caused by co-overexpression of dPDK1 and dAkt was reverted by using EMS mutagenesis, leading to three partial or complete loss-of-function mutations. dPDK1³ causes a G(352) to S substitution in the conserved DFG motif in the kinase subdomain VII. The D residue in this motif is essential for kinase activity by orienting the ATP-Mg²⁺ complex for phosphotransfer. dPDK1⁴ causes a P(441) to L substitution in a conserved residue in kinase subdomain VIII. In the dPDK1⁵ allele, a Q codon at position 437 in kinase subdomain VIII is mutated to a STOP codon. Because this latter mutation results in the formation of a truncated dPDK1 protein lacking part of the kinase domain and the Pleckstrin-homology domain, dPDK1⁵ is likely to be a null mutation. A fourth allele EP(3)3091 (dPDK1¹), from the Berkeley Drosophila Genome Project, has an EP element located in the third intron of dPDK1 and is homozygous lethal. It failed to complement dPDK1⁵, and the lethality was reversed by EP element excision (Rintelen, 2001).

Combinations of loss-of-function alleles provide mutants of varying strengths. Larvae homozygous for the dPDK1⁵ null allele or larvae of the dPDK1^1/5 heteroallelic combination die during the second instar stage. A less severe reduction in dPDK1 function (dPDK1^4/5) permits development of viable dPDK1 mutant flies that are delayed 1 day in development and smaller than their heterozygous siblings, having an 18% reduction in body weight. By measuring the cell density in the wing, the reduction in size and weight apparently is primarily caused by a decrease in cell size, because cell number is only slightly affected. The lethality associated with the dPDK1 null allele and the size defect of dPDK1 hypomorphs was rescued by ubiquitous expression of a wild-type dPDK1 transgene with armadillo (arm)-Gal4 as a driver. dPDK1^4/5 male flies are almost completely sterile, although they show no obvious defect in sperm morphology and motility and in mating behavior. That loss of zygotic dPDK1 function results in larval lethality is in contrast to a recent analysis of two dPDK1 mutations caused by the EP insertion EP(3)3091 (dPDK1¹) or a 10-kb deletion (dPDK1²), which are homozygous embryonic lethal. It is possible that the embryonic lethality observed by Cho (2001) is not caused by loss of dPDK1 function but by a linked lethal mutation on the same chromosome, because no rescue was attempted, and the phenotype was only analyzed in homozygotes. Consistent with this observation, larvae homozygous for a dPDK1¹ mutant chromosome, which has been cleaned from second hits by recombination, die during the second instar stage. Although it is very likely that dPDK1 functions during embryogenesis, like dAkt, maternal transcripts may be sufficient to support embryonic development (Rintelen, 2001).

To determine whether the effects of loss of dPDK1 function on cell growth and organ development are autonomous events, loss of dPDK1 was analyzed in clones of cells by using the FRT mitotic recombination system. In contrast to organism lethality, clones of cells homozygous for the dPDK1 null allele dPDK1⁵ survive to adulthood. These cells show no defect in their ability to differentiate into photoreceptor cells or accessory cells, but mutant photoreceptor cells are ~30% smaller than the heterozygous cells outside the clone, a strictly cell autonomous effect. To test whether an entire body part could develop in the absence of dPDK1 function, dPDK1 was selectively removed in much of the head primordium by using the ey-Flp system. Heads homozygous mutant for anyone of the three alleles, dPDK1³, dPDK1⁴, and dPDK1⁵, are reduced in size, which indicates that entire organs differentiate and develop in the absence of dPDK1 function, but that the final size of these organs autonomously depends on the amount of dPDK1 activity. The reduction in head size was most severe with dPDK1⁵ followed by dPDK1⁴ and dPDK1³, with the complete removal of dPDK1 function similar to that observed for loss-of-function mutations in the Drosophila insulin receptor (dInr), Dp110/PI(3)K, and dAkt (Rintelen, 2001).

The pronounced effect of loss of dPDK1 function on head size suggests that it is a dominant constituent in the dInr pathway. To test this possibility, the ability of complete and partial loss-of-function alleles of dPDK1 to reverse phenotypes caused by either overexpression of dInr or by mutations in dPTEN, the 3-phosphatidylinositide phosphatase, was evaluated. Overexpression of a wild-type dInr cDNA under the control of GMR-Gal4 leads to a marked increase in eye size and a slightly rough eye surface, an effect dominantly suppressed by removing one copy of dPDK1. Further reduction of dPDK1 function by the dPDK1^1/4 heteroallelic combination reduces the eye to almost wild-type size, suggesting that the amount of dPDK1 protein is rate-limiting for the dInr overgrowth phenotype. Null mutations in dPTEN cause lethality, and removal of dPTEN function in clones stimulates cell autonomous growth, suggesting that increased levels of PIP₃ promote growth and are the likely cause of lethality. Thus, if dPDK1 is an essential target of PIP₃, mutations in dPDK1 may suppress the dPTEN phenotype. Surprisingly, some dPTEN/dPDK1 double mutant flies survive to adulthood, indicating that the presumed PIP₃-induced lethality is primarily caused by the hyperactivation of dPDK1 or of one of its targets (Rintelen, 2001).

The fact that the growth phenotypes of dPDK1 mutations are similar to those caused by mutations in genes coding for dS6K, and dAkt, and that S6K1 is a mammalian PDK1 substrate, raises the possibility that dPDK1 may independently control growth through dS6K. This possibility was tested in the wing, which is composed of a dorsal and a ventral epithelial sheet that are tightly attached to each other through extracellular matrix. Selective overexpression of a wild-type dS6K cDNA in the dorsal wing epithelium with the apterous (ap)-Gal4 driver leads to a bending down of the wing blade, probably because of a cell-size increase in the dorsal surface. This phenotype is suppressed by a reduction of dPDK1 function. Although ap-Gal4 induced overexpression of wild-type dPDK1 alone had little effect on wing morphology, overexpression of a dPDK1^A467V variant is sufficient to cause a bent-wing phenotype. The corresponding amino acid substitution in the C. elegans PDK1 is thought to cause a hyperactivation of the kinase. The dPDK1^A467V-induced bent wing phenotype depends on normal levels of dS6K and dAkt, because null mutations in either of the corresponding genes dominantly suppress the phenotype. Together with the biochemical evidence in both cultured cells and in vivo, that dPDK1 controls the activity of dAkt and dS6K, these results provide functional evidence that dPDK1 is a key regulator in the control of growth and cell size by regulating the activity of two AGC kinases, dAkt and dS6K (Rintelen, 2001).

The effects of dPDK1 on dS6K raised the possibility that dPDK1 controls the activity of other AGC kinases in vivo, such as dRSK and dPKN, which have been implicated as mammalian PDK1 substrates. Because the developing eye depends on endogenous levels of dPDK1, whether lowering the dose of dPDK1 is sufficient to suppress dominantly the rough eye phenotype caused by overexpression of dRSK and dPKN under GMR-Gal4 control was tested. Reduction of dPDK1 activity in a viable dPDK1 mutant combination is sufficient to suppress the rough eye phenotype of dRSK but not of dPKN overexpression. These results suggest that at least in this in vivo assay, dRSK activity critically depends on dPDK1 function, whereas dPKN activity is not changed by a reduction in dPDK1 levels. This idea is in line with the recent finding that in PDK1^-/- embryonic stem cells the protein kinase C-related protein kinase PRK2 (CG2049), which shares extensive homology with PKN, is still partially phosphorylated at its T loop residue, indicating that PDK1-independent mechanisms may exist for the phosphorylation of the T loop of certain AGC kinases including dPKN (Rintelen, 2001).

These results show that dPDK1 is an essential component in the insulin signaling pathway in the control of cell growth and body size through its two substrates, dAkt and dS6K. These results are distinct from the genetic evidence in C. elegans where Akt is the primary target of PDK1 in dauer formation. Because mutations in the insulin signaling pathway do not show an autonomous alteration of cell size in C. elegans, the regulation of the rate of protein synthesis through S6K does not seem to be a primary target of this pathway. However, the fact that dPDK1 may yet have additional substrates is suggested by the genetic interaction with dRSK gain-of-function mutations and because viable dPDK1 males are almost completely sterile. Although mutations in components of the insulin signaling pathway such as dInr, chico, Dp110/PI(3)K, and dAkt cause female sterility, male sterility is not observed. Further genetic dissection of dPDK1 function is required to determine the role of dPDK1 in male fertility. These findings in Drosophila are consistent with the absence of insulin growth factor-1-induced activation of S6K, Akt, and RSK in mammalian PDK1^-/- embryonic stem cells, and therefore provide evidence for the functional conservation of branch points in kinase networks during evolution (Rintelen, 2001).

GENE STRUCTURE

A sequence ATCAGTT, which fits well to the consensus for transcription initiation in Drosophila ATCA(G/T)T(C/T) was found in the promoter region of DRAC-PK/Akt1, about 20 nucleotides from the 5'-end of cDNA SDE-RAC 109 (nucleotide 389). However, this sequence is not preceded by a typical TATA box, and the first one is present ~260 nucleotides upstream from the putative transcription initiation site. Analysis of the promoter region by primer extension using an oligonucleotide that hybridizes ~185 nucleotides downstream of the 5'-end of the SDE-RAC 109 cDNA reveals that transcription of the DRAC-PK gene initiates at four major sites (nucleotides 367, 378, 417, and 432) that all mapped in close proximity to the SDE-RAC 109 start site (Andjelkovic, 1995).

The first (noncoding) exon, located 1.2 kb upstream of exon 2, contains opa repeats, which are also present in several developmentally regulated Drosophila genes. The remaining six exons are coding and are separated by five small introns (60-70 base pairs long). The SDE-RAC 105 cDNA (which contains exons 2-7) has at its 5'-end 45 nucleotides derived from the 3'-end of the 1.2-kb-long intron and probably represents a splicing intermediate. The last exon encodes multiple polyadenylation signals. Analysis of the 3'-end of DRAC-PK cDNAs, isolated from a Drosophila embryo library, reveals that two of these are used (at positions 4374-4379 and 5590-5595). Also, four potential mRNA AUUUA destabilization signals are found in the 3`-untranslated region between the two polyadenylation signals. The same AUUUA sequences are present in the 3'-untranslated region of both human RAC-PKs, at positions 1709 and 1849 in the alpha isoform, and at position 1800 in the beta isoform sequence (Andjelkovic, 1995).

A putative initiation codon (AACCATG) in the correct context for translation initiation was found in the second exon. The predicted open reading frame encodes a 530-amino acid polypeptide highly homologous to human RAC-PKalpha and beta, with a predicted molecular mass of 59.9 kDa and an isoelectric point of 5.7. This reading frame remained open almost to the 5'-end of the second exon, but no upstream in-frame initiator codons could be found. Expression of the cDNA DRAC 7 in COS-1 cells produced a protein of the expected size (~66 kDa), but expression of the longer cDNAs SDE-RAC 109 and 105 has revealed the presence of a higher molecular weight form (~85 kDa) in addition to the major 66-kDa protein. These data suggest the existence of an upstream weaker initiation codon near the 5'-end of the second exon. Analysis of the genomic and cDNA sequences suggests that the putative initiator codon is an ACG preceded by CAAC, a sequence compatible with the consensus for translation initiation in Drosophila (C/A)AA(C/A). The open reading frame that starts from this upstream initiation codon translates into a 611 amino acid-long polypeptide, with a predicted molecular mass 68.5 kDa and an isoelectric point of 6.2. The apparent molecular mass on SDS-PAGE (~85 kDa) is higher than the predicted molecular mass, which could be explained by a high proline content (11%) in the N-terminal extension of the larger DRAC-PK polypeptide. The two protein forms were therefore termed DRAC-PK66 and DRAC-PK85, according to their apparent molecular masses on SDS-PAGE (Andjelkovic, 1995).

PROTEIN STRUCTURE

Amino Acids - 530 and 611

Structural Domains

The Akt proto-oncogene encodes a serine-threonine protein kinase whose carboxyterminal catalytic domain is closely related to the catalytic domains of all the known members of the protein kinase C (PKC) family. Akt, however, differs from PKC in its N-terminal region, which contains a domain related distantly to the SH2 domain of cytoplasmic tyrosine kinases and other signaling proteins, and which has been named the Akt homology (AH) domain. Low stringency hybridization of a c-akt AH probe to a panel of genomic DNAs from vertebrate and invertebrate eukaryotes detected multiple DNA bands (perhaps multiple genes) in all tested species. Drosophila DNA contains at least three hybridizing DNA bands. One of them was cloned, and found by sequence analysis, to define an Akt related gene (Dakt1). Comparison between the coding regions of c-akt and Dakt1 reveals 64.6% identity at the nucleotide level and 76.5% similarity at the amino acid level. The highest degree of homology is detected in the AH domain (68.3% similarity at the amino acid level) and the catalytic domain (83.3% similarity). Additional sequence comparisons reveal that the amino acid similarity between the catalytic domains of Dkt1 and the three known members of the Drosophila protein kinase C (PKC) family, Dpkc1, Dpkc2 and Dpkc3, is 68%, 63.6% and 67.1%, respectively. Dakt1 was mapped to Drosophila chromosome 3R 89BC. Its expression is subject to developmental regulation with the highest levels detected within the fourth hour of embryonic development. These results confirm that the AH domain of Akt defines new protein families in both vertebrate and invertebrate eucaryotes. The high degree of homology between the catalytic domains of Akt1 and the three known members of the Drosophila PKC family suggests an evolutionarily conserved functional relationship between the members of the two families (Franke, 1994).

The deduced amino acid sequences of the DRAC-PK/Akt1 proteins possess all conserved motifs of serine/threonine protein kinases. The motif -Gly-X-Gly-X-X-Gly-, residues 273-278 in the DRAC-PK85 sequence, with a Lys residue at position 295 conformed exactly to a consensus ATP binding motif. Two motifs, -Asp-Leu-Lys-Leu-Glu-Asn- and -Gly-Thr-Pro-Glu-Tyr-Leu-Ala-Pro-Glu-, both of which confer serine/threonine specificity, were found at amino acids 389-394 and 426-434, respectively (Andjelkovic, 1995).

DRAC-PK/Akt1 possess a PH domain that is located N-terminal to the catalytic domain and is 71% homologous to the PH domain of human RAC-PKs. This region has been identified in 71 signaling and cytoskeletal molecules. Two DRAC-PK polypeptides were detected, P66 and P85. DRAC-PKs has an extension at the amino-terminal region in comparison with human homologs. DRAC-PK85 polypeptide possesses an additional 81 amino acid residues at its N terminus that do not show any significant homology to sequences in the data bases. The predicted extension of DRAC-PK85 has an isoelectric point of 10.7 and is rich in serine/threonine residues. At the C terminus, both DRAC-PK forms have an 18-amino acid extension, which has a high serine/threonine content. The DRAC-PK catalytic domain shows the highest degree of homology to serum and glucocorticoid-regulated kinase (sgk; 72% homology, 57% identity); mitogen-stimulated ribosomal S6 kinase (69% homology), and the alpha catalytic subunit of bovine protein kinase A. DRAC-PKs has slightly lower homology to the Drosophila homologs of protein kinase C and protein kinase A. Of the three known Drosophila protein kinase C genes, DRAC-PK shows 72% homology to 98F and 62% to the Drosophila protein kinase A catalytic subunit DC0 (Andjelkovic, 1995).

date revised: 13 August 2000

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