Mutation of the PDK1 PH domain inhibits protein kinase B/Akt, leading to small size and insulin resistance

Abstract

PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.

FIG. 1.

Crystal structure of the isolated PDK1[K465E] PH domain. The structure was determined at a 1.80-Å resolution by molecular replacement and refined to a final R factor of 0.173 (R_free, 0.215). Further details on crystallization and statistics can be found in Table S1 in the supplemental material. (A) Comparison of the phosphoinositide-binding site of wild-type PDK1 (left) with that of the K465E mutant, mutation of which abrogates phosphoinositide binding (right). A stick representation of the interactions of Ins(1,3,4,5)P₄ (blue, inositol ring; purple/red, phosphate groups) with protein residues (green) in the phosphoinositide binding site of the PDK1-PH domain is shown. Lys465 is the central residue in the back of the pocket, and its mutation to Glu does not affect the overall structure of the PH domain but leads to reorganization of the phosphoinositide-binding site by attracting surrounding positively charged residues. The PDK1 PH K465E mutant was found to contain a sulfate molecule in the phosphoinositide-binding site derived from the crystallization buffer. (B) An electrostatic surface representation (from the GRASP software program [45]) of the phosphoinositide-binding site showing how mutation of Lys465 to Glu markedly alters both the shape and the basic nature of this pocket compared to the wild-type protein.

FIG. 2.

Generation of PDK1^K465E/K465E mice. (A) Diagram depicting the 3′ end of the endogenous PDK1 gene from exons 12 to 14, the targeting construct generated, the targeted allele with the neomycin selection cassette still present (NEO), and the targeted allele with the neomycin cassette removed by Cre recombinase. The black boxes represent exons, and the black triangles represent LoxP sites. Abbreviations: E, EcoRI; H, HindIII; S, SacI. The positions of the probes used for Southern analysis are shown as black bars. The knock-in allele containing the Lys465Glu mutation in exon 12 is marked with an asterisk and can be detected by genotyping using PCR primers K465E F and K465E R, which are depicted as arrows. (B) Genomic DNA purified from targeted ES cells from the indicated genotypes was digested with SacI and subjected to Southern analysis with the corresponding DNA probes. The wild-type allele generates an 18-kb fragment with both 5′ and 3′ probes, while the targeted allele give rise to a 6-kb fragment with the 5′ probe (left panel) and a 10-kb fragment with the 3′ probe (right panel). (C) The number (n) and proportion (%) of mice of each genotype resulting from heterozygous breeding are indicated. (D) Genomic DNA was PCR amplified with primers K465E F and K465E R. The wild-type (WT) allele produces a 196-bp fragment, while the knock-in allele generates a 236-bp product. The same DNA was subjected to PCR to generate a product that encompasses the knock-in mutation region in exon 12. The resultant PCR products were ligated onto the pCR-topo2.1 vector and transformed in E. coli and ∼30 independent clones were sequenced. The numbers of wild-type Lys465 and knock-in Glu465 sequences obtained for each genotype are indicated. (E) The upper diagram illustrates the mechanism by which PIF-Sepharose and PtdIns(3,4,5)P₃-agarose can be utilized to affinity purify PDK1. Mouse liver extracts or the PIF-Sepharose and PtdIns(3,4,5)P₃-agarose pull downs were subjected to immunoblot analysis with the indicated antibodies. (F) PDK1 was immunoprecipitated from liver extracts of the indicated genotypes and the activity assayed using the T308tide peptide as the substrate. Each point represents the mean activity ± standard error of the mean for three different samples with each assayed in triplicate.

FIG. 3.

Reduced size of PDK1^K465E/K465E mice. (A) The mean body weights of the indicated male and female mice are shown. Values represent the mean ± standard error of the mean for each data point obtained from no fewer than 20 mice per genotype. A representative photograph of indicated littermate 4- and 13-week-old male mice is shown. WT, wild type. (B) The organ volume of the indicated organs of PDK1^+/+ and PDK1^K465E/K465E littermates was measured from magnetic resonance imaging-obtained images or physical sections of fixed organs using the Cavalieri method as described in Materials and Methods. The data are represented as the means ± standard errors of the means for three different mice per genotype. (C) The relative cell size of the zona fasciculata cells of the adrenal glands of two PDK1^K465E/K465E mice compared to that for littermate PDK1^+/+ animals, which is given a value of 100%. Cell size was measured using the dissector principle as described in Materials and Methods.

FIG. 4.

PDK1^K465E/K465E mice are insulin resistant. (A and F) The indicated mice were deprived of food overnight and then injected intraperitoneally with glucose, and the blood glucose concentration was measured at the indicated times. (B and G) Normally fed mice were deprived of food for 2 h and then injected intraperitoneally with insulin, and the blood glucose concentration was measured at the indicated times. (C and H) Mice were left in the presence (Fed) or absence (Fasted) of food overnight, and the plasma insulin levels were measured for 13 (C) or 6 (H) mice of each genotype. (D and I) The indicated mice were fasted overnight and then allowed to refeed ad libitum for 1 or 6 h. Plasma insulin levels (upper panels) or blood glucose (lower panels) were measured for 6 mice per genotype at the indicated times. The data are presented as the means ± standard errors of the means for each data point. (E) A hyperinsulinemic euglycemic clamp study was performed as described in Materials and Methods using six littermate mice of each genotype of 3 to 5 months of age. The hyperinsulinemic study started with a bolus of insulin (100 mU/kg), followed by continuous infusion (3.5 mU/kg/min). A variable infusion of 12.5% d-glucose solution was adjusted to maintain euglycemia, as measured via tail bleeding. The data are presented as the mean ± standard error of the mean for each data point.

FIG. 5.

Islet volume in young and old PDK1^K465E/K465E animals. The total volume of the pancreas and islet cells of the indicated mice was measured using the unbiased Cavalieri method. The pancreas volume (left), the islet volume (middle), and the percentage of pancreas volume that is occupied by endocrine pancreas (right) are represented as the means ± standard errors of the means for three different male mice per genotype of age 12 to 6 weeks (A) or 84 to 88 weeks (B). WT, wild type; V/V, vol/vol.

FIG. 6.

Activation and phosphorylation of PKB in PDK1^K465E/K465E mice and ES cells. (A to D) Mice were fasted overnight and intravenously injected with either saline (for the 0-min time point control) or 0.5 mU/g of insulin. Skeletal muscle (A), heart (B), liver (C), or adipose tissue (D) at the indicated time points was rapidly extracted and frozen in liquid nitrogen. (E) The indicated ES cell lines were grown to 80% confluence, serum starved for 4 h, and then either left unstimulated or stimulated with 20 ng/ml IGF1 for the indicated times. Left panels, PKBα or PKBβ (in liver or adipose tissue) was immunoprecipitated and assayed using the Crosstide peptide. Each point represents the mean activity ± standard error of the mean for samples derived from three different mice, with each sample assayed in triplicate. WT, wild type. Right panels, the cell lysates from skeletal muscle (A), heart (B), liver (C), adipose tissue (D), or ES cells (E) were immunoblotted with the indicated antibodies, and each lane represents a sample derived from a different mouse or plate.

FIG. 7.

Activation of S6K1, SGK1, and RSK in PDK1^K465E/K465E mice. Mice were fasted overnight and then either intravenously injected with saline (for the 0-min time point control), injected with 0.5 mU/g of insulin (A, C, and D), or allowed to refeed ad libitum (B). At the indicated time points, cardiac muscle (A and D) or liver (B and C) were rapidly extracted and frozen in liquid nitrogen. S6K1 (A and B) or RSK isoforms (D) were immunoprecipitated from the indicated extracts and their activity assayed using Crosstide. Each point represents the mean activity ± standard error of the mean for samples derived from three different mice, with each sample assayed in triplicate. WT, wild type. (C) SGK1 was immunoprecipitated from the livers of littermate PDK1^+/+ and PDK1^K465E/K465E mice (upper panel) as well as SGK1^+/+ and SGK1^−/− mice (60) (lower panel). Its activity was assessed by measuring phosphorylation of NDRG1, followed by immunoblot analysis employing a phospho-specific antibody recognizing the phosphorylation sites targeted by SGK1 (termed Tx3-P). The indicated cell extracts were also immunoblotted with the indicated antibodies, and each lane represents a sample derived from a different mouse.