In yeast producing PIP3, PKB/c-Akt localizes to the plasma membrane and its phosphorylation is enhanced

In yeast producing PIP3, PKB/c-Akt localizes to the plasma membrane and its phosphorylation is enhanced. Thr308 phosphorylation, but not Ser473 phosphorylation, requires the yeast orthologues of mammalian PDK1 (3-phosphoinositide-dependent protein kinase-1): Pkh1 and Pkh2. Elimination of yeast Tor1 and Tor2 function, or of the related kinases (Tel1, Mec1 and Tra1), Rhein-8-O-beta-D-glucopyranoside did not block Ser473 phosphorylation, implicating another kinase(s). Reconstruction of the PI3K/PTEN/Akt pathway in yeast permits incisive study of these enzymes and analysis of their functional interactions in a simplified context, establishes a new tool to screen for novel agonists and antagonists and provides a method to deplete PIP2 uniquely in the yeast cell. genome encodes: (i) two functional PDK1 orthologues (Pkh1 and Pkh2) involved in cell integrity and endocytosis [16,17]; (ii) an apparent PTEN orthologue (Tep1) of uncharacterized biological function [18,19]; (iii) an Akt-like protein kinase (Sch9), which lacks an apparent PH domain, involved in nutrient sensing, ribosome biogenesis, lifespan and cell-size control [20]; and (iv) clear-cut homologues of the PIKK family, specifically Tor1 and Tor2 (mTOR) [21], Tel1 (ATM) [22], Mec1 (ATR) [23] and Tra1 (most resembles DNA-PKcs) [24]. To address central questions in the biology of PIP3-dependent signalling and to establish a readily accessible and versatile tool to screen for pharmacological agents that influence this critically important pathway, we devised methods to successfully reconstitute the mammalian PI3K/PTEN/Akt pathway in yeast cells, which is described here. conversion of the essential PIP2 pool into PIP3 by expression of PI3K impaired yeast growth by altering morphogenesis and vesicular trafficking. The function of PTEN could be readily assessed by its ability to reverse the growth inhibition caused by PI3K. PIP3 generation led to membrane translocation and activation of Akt, enhancing its phosphorylation at both Thr308 and Ser473. The yeast PDK1 orthologues are required for PDK1 site phosphorylation, whereas none of the yeast PIKK family members seems necessary for PDK2 site phosphorylation, implicating some other endogenous enzyme. EXPERIMENTAL Strains, media and growth conditions The strains used in the present study are listed in Table 1. DH5 F[K12((strains used in the present study YCplac111(and yeast and other basic molecular biology methods were carried out using standard procedures. To generate plasmid YCpLG-PI3K, the cDNA encoding PI3K-CAAX was excised from plasmid Psg5/5MycTp110XCAAX [25] (a gift from M. Collado, Spanish National Cancer Centre, Madrid, Spain) with BamHI and cloned into the same site in yeast vector YCpLG [26]. To produce plasmid YCpLG-PI3KK802R (where K802R stands for Lys802Arg), bearing a catalytically inactive Rhein-8-O-beta-D-glucopyranoside (kinase-dead) allele of PI3K-CAAX, site-directed mutagenesis was carried out using a DpnI-based strategy [27] with Turbo PfuI DNA polymerase (Stratagene) and the primers 5-CAGAACAATGAGATCATCTTTCGAAATGGGGATGATTTACGGC-3 and 5-GCCGTAAATCATCCCCATTTCGAAAGATGATCTCATTGTTCTG-3. Rhein-8-O-beta-D-glucopyranoside Cassettes in which cDNAs encoding either c-Akt, c-AktK179M or an N-myristoylated c-Akt were fused in frame to an HA (haemagglutinin) epitope-tagged version of eGFP (enhanced green fluorescence Rhein-8-O-beta-D-glucopyranoside protein) [HACeGFP-Akt, HACeGFP-AktK179M and myr-HACeGFP-Akt respectively] were excised with HindIII and BamHI from the original Pcefl(X)-derived plasmids that were constructed for expression in mammalian cells [28] (a gift from M. Lorenzo, Universidad Complutense, Madrid, Spain) and cloned into the corresponding sites in yeast vector pYES2 (Invitrogen), yielding plasmids pYES-GFP-c-Akt, pYES-GFP-c-AktK179M and pYES-myr-GFP-c-Akt respectively. The cDNA encoding PTEN was excised with EcoRI from plasmid Pcmvpten [29] [a gift from J.M. Paramio, CIEMAT (Centro de Investigaciones Energticas, Medioambientales y Tecnolgicas, Madrid, Spain)] and inserted into the same site in pYES2, generating pYES-PTEN. Plasmid pYES-PTENG129D, expressing a catalytically inactive (phosphatase-dead) allele, was generated by site-directed mutagenesis, as above, but using primers 5-CACTGTAAAGCTGGAAAGGAACGAACTGGTGTAATG-3 (upper) and 5-CATTACACCAGTTCGTTCCTTTCCAGCTTTACAGTG-3 (lower). To construct plasmid pYES-Tep1-Myc, first the coding sequence was amplified by PCR from yeast genomic DNA using the primers 5-CGGATCCATGAGAGAGGAGGGGAGTG-3 (upper) and 5-GGGATCCTATAATTTCCCATTCCAAT-3 (lower) and Mouse monoclonal to 4E-BP1 cloned into the BamHI site in a yeast vector, pRS306-Myc6, which had been previously generated by Rhein-8-O-beta-D-glucopyranoside inserting a Myc6 epitope into the polylinker in the integrative vector, pRS306 [30]. Secondly, the resulting chimaera, we used a now-standard PCR-based method [31] in which was amplified using the upper primer indicated above, was amplified with the lower primer indicated above, with the following primers to generate the desired junction: 5-GCCTCATTAGAAATTCCTGGTCTATAATCCAGAT-3 and 5-ATCTGGATTATAGACCAGGAATTTCTAATGAGGC-3. To construct a plasmid expressing a Tep1CGFP fusion, the gene was amplified by PCR.