Characterization of the Met326Ile variant of phosphatidylinositol 3-kinase p85alpha

Abstract

Phosphatidylinositol 3-kinase is a key step in the metabolic actions of insulin. Two amino acid substitutions have been identified in the gene for the regulatory subunit of human p85alpha, Met-326Ile, and Asn-330Asp, and the former has been associated with alterations in glucose/insulin homeostasis. When the four human p85alpha proteins were expressed in yeast, a 27% decrease occurred in the level of protein expression of p85alpha(Ile/Asp) (P = 0.03) and a 43% decrease in p85alpha(Ile/Asn) (P = 0.08) as compared with p85alpha(Met/Asp). Both p85alpha(Ile/Asp) and p85alpha(Ile/Asn) also exhibited increased binding to phospho-insulin receptor substrate-1 by 41% and 83%, respectively (P < 0.001), as compared with p85alpha(Met/Asp). The expression of p85alpha(Ile) was also slightly decreased and the binding to insulin receptor substrate-1 slightly increased in brown preadipocytes derived from p85alpha knockout mice. Both p85alpha(Met) and p85alpha(Ile) had similar effects on AKT activity and were able to reconstitute differentiation of the preadipocytes, although the triglyceride concentration in fully differentiated adipocytes and insulin-stimulated 2-deoxyglucose uptake were slightly lower than in adipocytes expressing p85alpha(Met). Thus, the Met-326Ile variant of p85alpha is functional for intracellular signaling and adipocyte differentiation but has small alterations in protein expression and activity that could play a role in modifying insulin action.

Figure 1

Schematic alignment of the structural features of p85α, AS53 (or p55α), p50α and p55^PIK (or p55γ). AS53 and p50α are 100% homologous to p85α except for the N-terminal 34 and 6 amino acids (AA) which are unique. P55^PIK is the product of a separate gene and is ≈70% identical with p85α at the nucleotide level in the two SH2 domains and the p110-binding region and 44% identical in the N-terminal 34 amino acids (3). The methionine at codon 326 in p85α is conserved among species (human, bovine, rat, and mouse; ref. 12) suggesting a potentially important role of this residue for the function of the protein. A valine is present in human p55^PIK at the corresponding position to Met-326. The numbers below p85α indicate the amino acid positions.

Figure 2

(A) Expression of human p85α in yeast. The yeast strain L40 was cotransformed with pACTII/IRS-1 and pVJL-HIR/p85α encoding the indicated combination of amino acids at codons 326 and 330. Data represent the average protein levels of p85α from four independent experiments, and error bars indicate the SE (Met/Asp 100%, Ile/Asp 73 ± 9%, Met/Asn 100 ± 12%, Ile/Asn 57 ± 16%). The bar indicated by * is significantly decreased as compared with p85α^Met/Asp (P = 0.03). (Met = M, Ile = I, Asp = D, and Asn = N). (B) Interaction between p85α and IRS-1 in the yeast two-hybrid system. The β-galactosidase assay was performed in the presence (black) [no expression of insulin receptor β-subunit (IRβ)] or absence (white) (expression of IRβ) of L-methionine. The bars represent the β-galactosidase activity corrected for protein expression levels. Data represent 12 independent experiments, each in triplicate, and are expressed as percentage of p85α^Met/Asp (activity during IRβ expression: Met/Asp 100%, Ile/Asp 141 ± 4%, Met/Asn 107 ± 4%, Ile/Asn 183 ± 8%). The values are means ± SE. Bars indicated by * are significantly increased as compared with p85α^Met/Asp (P < 0.001). (C) Expression of human p85α in brown preadipocytes. Brown preadipocytes derived from p85α knockout mice were transfected with human p85α cDNA encoding the indicated combination of amino acids at codons 326 and 330. Data represent the average protein levels of p85α from three experiments performed in duplicate on four independent clones expressing p85^Met/Asp and p85^Ile/Asp, respectively. Error bars indicate the SE (Met/Asp 100%, Ile/Asp 86 ± 10%). (D) Interaction with IRS-1 in brown preadipocytes. Bars represent the interaction with IRS-1 after adjusting for protein expression at basal (black) or after insulin stimulation (white) (Insulin stimulated interaction: Met/Asp 100%, Ile/Asp 114 ± 13%). The experiments were performed three times in duplicate on four independent clones from each cell line (Met/Asp and Ile/Asp). Error bars indicate the SE.

Figure 3

Triglyceride concentration during differentiation. The bars represent triglyceride concentrations measured 1 (gray), 3 (white), and 6 (black) days after induction of the cells. [Triglyceride concentration (μg triglyceride/mg protein) at day 6: WT 163 ± 28, KO 68 ± 16, Met/Asp 163 ± 34, Ile/Asp 119 ± 19).] The differentiation was performed three times and error bars indicate the SE.

Figure 4

Insulin-stimulated glucose uptake. Differentiated brown adipocytes were serum starved for 16–18 h; insulin was added at the indicated concentrations. Data are expressed as fold glucose uptake from the basal glucose uptake. The graphs present four independent experiments, and error bars indicate the SE.