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. 2010 Mar 15;123(Pt 6):983-92.
doi: 10.1242/jcs.064022.

Chemotactic activation of Dictyostelium AGC-family kinases AKT and PKBR1 requires separate but coordinated functions of PDK1 and TORC2

Xin-Hua Liao  1 , Jonathan Buggey, Alan R Kimmel
Affiliations

Chemotactic activation of Dictyostelium AGC-family kinases AKT and PKBR1 requires separate but coordinated functions of PDK1 and TORC2

Xin-Hua Liao et al. J Cell Sci. .

Abstract

Protein kinases AKT and PKBR1 of Dictyostelium belong to the AGC protein kinase superfamily. AKT and PKBR1 are phosphorylated at similar sites by phosphoinositide-dependent kinase 1 (PDK1) and TORC2 kinases; however, they have different subcellular localizing domains. AKT has a phosphoinositide 3-kinase (PI3K)/phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)]-regulated PH (pleckstrin homology) domain whereas PKBR1 is myristoylated and persistently membrane localized. Using strains defective for PI3K/PtdIns(3,4,5)P(3)-, PDK1- and TORC2-signaling or strains that express phospho-site mutants of AKT and PKBR1, we dissect the different roles of PI3K/PtdIns(3,4,5)P(3), PDK1 and TORC2. We show that activation of AKT and PKBR1 requires PDK1-site phosphorylation, but that phosphorylation by TORC2 is insufficient for AKT or PKBR1 activation. However, PDK1-site phosphorylation is dependent on phosphorylation by TORC2, which suggests that there is regulatory coordination among PDK1, TORC2 and their phospho-site targets. This defines a separate input for signaling in control of chemotaxis and dependency on PDK1 function. We also demonstrate that PDK1 in Dictyostelium functions independently of PI3K/PtdIns(3,4,5)P(3). Finally, we show that AKT and PKBR1 exhibit substrate selectivity and identify two novel lipid-interacting proteins preferentially phosphorylated by AKT. Despite certain similarities, AKT and PKBR1 have distinct regulatory paths that impact activation and effector targeting, with PDK1 serving a central role.

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Figures

Fig. 1.
Fig. 1.
AKT and PKBR1 are activated by chemoattractant stimulation with cAMP and folate and phosphorylate preferential substrates. (A) Generic diagram of AKT and PKBR1 with phosphorylated threonine at PDK1 and PDK2/HM sites. (B-E) WT, akt−/pkbR1−, akt−, and pkbR1− cells were collected seconds (S) following stimulation with cAMP or folate and assayed by immunoblot with α-phospho-PDK1 site (p-PDK1), α-phospho-PDK2 site (p-PDK2), α-actin, and/or α-AKT substrate motif (p-Substrate). Wildtype (WT) substrate bands are indicated. P78 and P53 are preferentially phosphorylated by AKT. P65 is preferentially phosphorylated by PKBR1.
Fig. 2.
Fig. 2.
AKT and PKBR1 are differentially sensitive to regulation by TORC2. (A-C) WT, rictor−(pia−), and SIN1−(rip3−) cells were collected following stimulation with cAMP or folate and assayed by immunoblot with α-phospho-PDK1 site (p-PDK1), α-phospho-PDK2 site (p-PDK2), α-actin and α-AKT substrate motif (p-Substrate).
Fig. 3.
Fig. 3.
AKT and PKBR1 are differentially regulated by PI3K signaling. (A-C) WT, pi3k1—6−, pi3k1-5−, pi3k1-5−/pten− and pten− cells were collected following stimulation with cAMP or folate and assayed by immunoblot with α-phospho-PDK1 site (p-PDK1), α-phospho-PDK2 site (p-PDK2), α-actin and α-AKT substrate motif (p-Substrate). (D) Differential effects of LY on PDK1/2 phosphorylation of AKT and PKBR1. WT cells were pre-treated with various doses of LY and collected 15 seconds following stimulation with folate; they were assayed by immunoblot with α-phospho-PDK1 site (p-PDK1), α-phospho-PDK2 site (p-PDK2) and α-actin. Band phosphorylations were quantified and normalized to 1 for 0 μM LY.
Fig. 4.
Fig. 4.
Dictyostelium PDK1 regulates AKT and PKBR1, and chemotaxis and development independently of PtdIns(3,4,5)P3. (A) WT, pdkA−, pdkB− and pdkA/B− cells were collected following stimulation with cAMP or folate and assayed by immunoblot with α-phospho-PDK1 (p-PDK1), α-phospho-PDK2 site (p-PDK2), α-actin and α-AKT substrate motif (p-Substrate). (B) WT and pdkA− cells were plated in submerged culture at 1×105 cells/cm2 and imaged after 8 hours. (C) WT, pdkA−, pdkB− and pdkA/B− cells were plated on solid substrata and developed for 24 hours; arrows indicate sori of terminally differentiated organisms. (D) Lipid binding assay for PdkA. Cell extracts from pdkA-nulls expressing functional GFP-PdkA were incubated with a PIP lipid strip, as indicated. Bound proteins were detected with α-GFP. WT cells expressing GFP or GFP fused to CRAC were controls. Relative expression was determined by immunoblot with α-GFP. (E) PI3K, PDK1 and TORC2 regulation of AKT and PKBR1. PI3K regulates PtdIns(3,4,5)P3 accumulation and recruitment of AKT to membranes. Membrane-bound AKT and PKBR1 are phosphorylated by PDK1 and TORC2 independently of PI3K.
Fig. 5.
Fig. 5.
PDK2/HM phosphorylation of PKBR1 and AKT is not sufficient for activation by cAMP. (A) pkbR1− cells transfected with control vector or vectors engineered to express PKBR1WT, PKBR1T309A, or PKBR1T470A and collected following stimulation with cAMP or folate and assayed by immunoblot with α-phospho-PDK1 (p-PDK1), α-phospho-PDK2 site (p-PDK2), α-actin, and α-AKT substrate motif (p-Substrate). (B) The various pkbR1− strains (see Fig. 5A) were plated on solid substrata and developed for 24 hours; arrows indicate terminally differentiated organisms. (C) akt− cells transfected with control vector or vectors engineered to express AKTWT, AKTT278A, or AKTT435A and collected following stimulation with either cAMP or folate and assayed by immunoblot with α-phospho-PDK1 (p-PDK1), α-phospho-PDK2 site (p-PDK2), α-actin and α-AKT substrate motif (p-Substrate). (D) A generic diagram of AGC kinases AKT and PKBR1 indicating the coordination of kinases PDK1 and TORC2 to phosphorylate the threonine residues in the kinase and C-terminal domains, respectively, at PDK1 and PDK2/HM sites.
Fig. 6.
Fig. 6.
Purification and identification of AKT preferential substrates PHAPS and SHAPS. (A) akt− and rip3− cells were lysed 15 seconds after folate stimulation. Samples from cell lysates and AKT-substrate immunoprecipitations (IP) were immunoblotted with α-phospho-AKT substrate. (B) Confirmation of P78 and P53 as AKT substrates. The TAP epitope was fused to the endogenous P78 and P53 gene loci by targeted homologous recombination; there are two P78 genes in Dictyostelium (supplementary material Fig. S2) and only 1 P53 gene. Equivalent cell sample volumes of WT, P78-TAP or P53-TAP cells were collected at various times following stimulation with folate and assayed by immunoblot with α-phospho-AKT substrate and α-TAP. A new band detected by both antibodies is indicated by an arrow in each TAP cell and is absent in WT cells. P53 is detected only in WT cells. (C) Structure of PHAPS and SHAPS. PHAPS (P78) contains SAM and PH domains and the phosphorylated AKT motif KKRTTpT. SHAPS (P53) contains BAR and SH3 domains.
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