The life cycle of Dictyostelium consists of distinct growth and developmental phases. During development, starving cells first aggregate by chemotaxis, and then undergo cell differentiation and morphogenesis forming fruiting bodies consisting of stalks bearing on topa mass of spores. Development is regulated by cAMP, which functions as chemoattractant, extracellular hormone-like signal and intracellular second messenger. Cyclic cAMP is produced by three structurally different adenylyl cyclases, ACA, ACG and ACB, which have distinctive, but partially overlapping patterns of expression. During the first hours of starvation, cAMP is secreted and acts by binding to the serpentine receptor cAR1, triggering chemotactic cell movement and stimulating the adenylyl cyclase A (ACA) through the Ga2bg protein, thus activating a signal relaying feedback while concomitantly leading to developmental gene expression by PKA activation. G protein-dependent ACA activation requires the concomitant activity of two cytosolic proteins, Crac and PIA. PIA is the ortholog of Rictor (thus named Pia/Rictor), a subunit of the target of rapamycin complex 2 (TORC2), together with the serine-threonine kinase TOR, Lst8 and Rip3. TORC2 is also responsible for the phosphorylation of AKT/PKBs (PKBR1 and AKT). When cells are stimulated with cAMP, PKBR1 and AKT are rapidly and transiently phosphorylated within their hydrophobic motifs (HMs) and activation loops (ALs), as it has been described for their mammalian counterparts. In AKT, AL phosphorylation also requires the recruitment of the enzyme to the membrane by transient accumulation of PIP3. Because AL phosphorylation in PKBR1 is independent of PIP3, the mechanism of its regulation is not known. The TORC2-PKBs pathway is activated at the cell leading edge, where it regulates actin recruitment, and thus cell polarization during chemotaxis. Homologs of these proteins also function in metazoan chemotaxis, hence the importance of Dictyostelium as a model organism for studying the molecular mechanisms that regulate chemotaxis and development. To investigate the intricate signalling network that mediate cAMP signalling, we have exploited near-saturation mutagenesis by restriction enzyme mediated integration (REMI) to generate suppressors of a Dictyostelium mutant unable to undergo chemotaxis and aggregation. This mutant, named HSB1, is characterized by a point mutation in the piaA/rictor gene, resulting in a single amino acid exchange (G917D). Both in vivo and in vitro G protein-linked adenylyl cyclase activation is defective in HSB1. Transfection with the gene encoding the cytosolic regulator PIA rescued the mutant phenotype. When overexpressed in the wild-type background, Pia/RictorG917D displayed a partial dominant-negative effect, probably competing with wild-type Pia/Rictor, via the non-mutated domain, thus delaying development.

INACTIVATION OF A NOVEL HECT E3 UBIQUITIN LIGASE IN A PIA/RICTOR DEFICIENT DICTYOSTELIUM MUTANT RESTORES CHEMOTAXIS AND DEVELOPMENT

PERGOLIZZI, Barbara;BRACCO, Enrico;BOZZARO, Salvatore
2016

Abstract

The life cycle of Dictyostelium consists of distinct growth and developmental phases. During development, starving cells first aggregate by chemotaxis, and then undergo cell differentiation and morphogenesis forming fruiting bodies consisting of stalks bearing on topa mass of spores. Development is regulated by cAMP, which functions as chemoattractant, extracellular hormone-like signal and intracellular second messenger. Cyclic cAMP is produced by three structurally different adenylyl cyclases, ACA, ACG and ACB, which have distinctive, but partially overlapping patterns of expression. During the first hours of starvation, cAMP is secreted and acts by binding to the serpentine receptor cAR1, triggering chemotactic cell movement and stimulating the adenylyl cyclase A (ACA) through the Ga2bg protein, thus activating a signal relaying feedback while concomitantly leading to developmental gene expression by PKA activation. G protein-dependent ACA activation requires the concomitant activity of two cytosolic proteins, Crac and PIA. PIA is the ortholog of Rictor (thus named Pia/Rictor), a subunit of the target of rapamycin complex 2 (TORC2), together with the serine-threonine kinase TOR, Lst8 and Rip3. TORC2 is also responsible for the phosphorylation of AKT/PKBs (PKBR1 and AKT). When cells are stimulated with cAMP, PKBR1 and AKT are rapidly and transiently phosphorylated within their hydrophobic motifs (HMs) and activation loops (ALs), as it has been described for their mammalian counterparts. In AKT, AL phosphorylation also requires the recruitment of the enzyme to the membrane by transient accumulation of PIP3. Because AL phosphorylation in PKBR1 is independent of PIP3, the mechanism of its regulation is not known. The TORC2-PKBs pathway is activated at the cell leading edge, where it regulates actin recruitment, and thus cell polarization during chemotaxis. Homologs of these proteins also function in metazoan chemotaxis, hence the importance of Dictyostelium as a model organism for studying the molecular mechanisms that regulate chemotaxis and development. To investigate the intricate signalling network that mediate cAMP signalling, we have exploited near-saturation mutagenesis by restriction enzyme mediated integration (REMI) to generate suppressors of a Dictyostelium mutant unable to undergo chemotaxis and aggregation. This mutant, named HSB1, is characterized by a point mutation in the piaA/rictor gene, resulting in a single amino acid exchange (G917D). Both in vivo and in vitro G protein-linked adenylyl cyclase activation is defective in HSB1. Transfection with the gene encoding the cytosolic regulator PIA rescued the mutant phenotype. When overexpressed in the wild-type background, Pia/RictorG917D displayed a partial dominant-negative effect, probably competing with wild-type Pia/Rictor, via the non-mutated domain, thus delaying development.
12th Internation Congress of Cell Biology
Praga
21-25 Luglio
12th Internation Congress of Cell Biology
250
250
Barbara, Pergolizzi; Enrico, Bracco; Salvatore, Bozzaro
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/1621675
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