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).

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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