A Protein with Similarity to PTEN Regulates Aggregation Territory Size by Decreasing Cyclic AMP Pulse Size during Dictyostelium discoideum Development

Abstract

An interesting but largely unanswered biological question is how eukaryotic organisms regulate the size of multicellular tissues. During development, a lawn of Dictyostelium cells breaks up into territories, and within the territories the cells aggregate in dendritic streams to form groups of ϳ20,000 cells. Using random insertional mutagenesis to search for genes involved in group size regulation, we found that an insertion in the cnrN gene affects group size. Cells lacking CnrN (cnrN ؊ ) form abnormally small groups, which can be rescued by the expression of exogenous CnrN. Relayed pulses of extracellular cyclic AMP (cAMP) direct cells to aggregate by chemotaxis to form aggregation territories and streams. cnrN ؊ cells overaccumulate cAMP during development and form small territories. Decreasing the cAMP pulse size by treating cnrN ؊ cells with cAMP phosphodiesterase or starving cnrN ؊ cells at a low density rescues the small-territory phenotype. The predicted CnrN sequence has similarity to phosphatase and tensin homolog (PTEN), which in Dictyostelium inhibits cAMP-stimulated phosphatidylinositol 3-kinase signaling pathways. CnrN inhibits cAMP-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation, Akt activation, actin polymerization, and cAMP production. Our results suggest that CnrN is a protein with some similarities to PTEN and that it regulates cAMP signal transduction to regulate territory size.

Extracellular polyphosphate signals through Ras and Akt to prime Dictyostelium discoideum cells for development

“…The cells used were Dictyostelium wild-type Ax2, pakD − (DBS0350281) (Garcia et al, 2014), erk1 − (DBS0350622) (Gaskins et al, 1994), pkbA − (DBS0349876) (Meili et al, 1999), pkbA − /pkgB − (DBS0236785) (Meili et al, 2000), rasC − (DBS0236853) (Daniel et al, 1994), napA − (DBS0236596) (Ibarra et al, 2006), adrm1 − (DBS0238203) (Cherix et al, 2006), fslB − (DBS0350230) (Prabhu and Eichinger, 2006), fslK − (DBS0350229) (Prabhu and Eichinger, 2006), ctnA − (DBS0235624) (Brock and Gomer, 1999), i6kA − (DBS0236426) (Luo et al, 2003), grlB − (DBS0350074) (Prabhu and Eichinger, 2006), grlE − (DBS0350075) (Prabhu and Eichinger, 2006), grlH − (DBS0350226) (Prabhu and Eichinger, 2006), cfaD − (DBS0302444) (Bakthavatsalam et al, 2008), lst8 − (DBS0236517) (Lee et al, 2005), ppk1 − (DBS0350685) (Livermore et al, 2016) and cnrN − (DBS0302655) (Tang and Gomer, 2008). Mutant genotypes were verified by PCR, and cells were kept under constant selection.…”

“…Cells lacking RasC (Daniel et al, 1994), the p21-activated kinase PakD (Garcia et al, 2014), Erk1 (also known as ErkA) (Gaskins et al, 1994), the Dictyostelium Akt homologs Pkba and PkgB (Meili et al, 1999(Meili et al, , 2000, and the suppressor of cAMP receptor (SCAR) component NapA (Ibarra et al, 2006) were all nonresponsive to polyphosphate-induced development (Table 3). Cells lacking the secreted factor CtnA (Brock and Gomer, 1999), the frizzled-like receptors FslB and FslK (Prabhu and Eichinger, 2006), the inositol hexakisphosphate kinase I6kA (Luo et al, 2003), the G-protein coupled receptors GrlB, GrlE and GrlH (Prabhu and Eichinger, 2006) the autocrine factor CfaD (Bakthavatsalam et al, 2008), the target of rapamycin complex 2 (TORC2) component Lst8 (Lee et al, 2005), the Dictyostelium polyphosphate kinase Ppk1 (Livermore et al, 2016), and the PTEN homolog CnrN (Tang and Gomer, 2008) did form aggregates in response to polyphosphate (Table 3). Of the transformants that were unresponsive to polyphosphate, cells lacking RasC, PakD and Erk1 were unresponsive to Mg132-induced development while cells lacking Akt (i.e.…”

A Dictyostelium Secreted Factor Requires a PTEN-Like Phosphatase to Slow Proliferation and Induce Chemorepulsion

“…CnrN is a PTEN-like protein in Dictyostelium that has PTEN-like phosphatase activity [20], [21]. In the absence of CnrN, levels of PIP 3 are higher than in wild-type cells [20].…”

“…In the absence of CnrN, levels of PIP 3 are higher than in wild-type cells [20]. Akt, a downstream target in PI3K pathways, usually requires translocation and phosphorylation from the cytosol to the membrane for its activation [22], [23], [24].…”

“…Akt, a downstream target in PI3K pathways, usually requires translocation and phosphorylation from the cytosol to the membrane for its activation [22], [23], [24]. During development, Akt translocation and phosphorylation is increased in the absence of CnrN [20]. The increases in Akt translocation, Akt phosphorylation, and levels of PIP 3 in cnrN¯ cells suggests that CnrN acts as a negative regulator of PIP 3 and Akt, which are both components of PI3K pathways [20], [24].…”