A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome

Abstract

Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

Copyright © 2015 Elsevier Inc. All rights reserved.

Figures

Figure 1

GLUT1 is phosphorylated in vivo and in vitro. A) The central cytoplasmic loop of GLUT1 (Loop 6) is phosphorylated by PKCβ1 and PKCδ in vitro. B) Sequence of vertebrate homologs of GLUT1 show conservation of several residues surrounding S226 when compared to optimal PKC consensus phosphorylation motifs. Colors (blue, basic; purple, serine; green, hydrophobic) highlight conserved residues. C) HeLa cell extract phosphorylates GST-GLUT1-Loop6 but this phosphorylation can be efficiently inhibited by a PKC inhibitor (Gö-6983). D) HA-GLUT1 purified from TPA-treated Rat2 fibroblasts were trypsin digested and analyzed by Q-exactive mass spectrometry. Labeled N-terminal (red) and C-terminal (blue) peaks unambiguously confirm phosphorylation of S226. Many peptides show neutral loss of phosphoric acid (−97.98/z), which is also consistent with phosphorylation at S226. E) PKCβ1 phosphorylates full length HA-GLUT1 in vitro but not in the presence of Gö-6983. Asterisk indicates a non-specific band. F) Phosphorylation of WT GLUT1 is induced by TPA and inhibited by PKC inhibitors, Rö-31-8220 and Gö-6983. The pGLUT1 S226 blot was stripped and reprobed for Actin. See also Figure S1, S2, and Table S1.

Figure 2

GLUT1 S226A is impaired in its response to TPA. A) WT, but not S226A, GLUT1 is specifically phosphorylated on S226 after treatment with TPA in Rat2 cells. pGLUT1 S226 blot was stripped and reprobed for Actin. B) Rat2 fibroblasts expressing WT, but not S226A, GLUT1 increase 2DG uptake within 30 minutes of treatment by TPA. Data fit against a three parameter dose response curve. (n=6 independent experiments, each performed in triplicate, error bars = SD). C) Western blot from membranes of 10 oocytes injected with the indicated in vitro transcribed mRNA or water (2 days) followed by treatment with TPA (20 min) demonstrates that the WT GLUT1 is phosphorylated after TPA treatment. D) Immunoflourescent images from oocytes injected with the indicated mRNA (bottom) followed by staining with the indicated antibody (top) with or without TPA treatment demonstrate comparable expression and trafficking of WT and 226A transporter to the cell membrane. Phosphorylated GLUT1 is only detected after TPA treatment of WT GLUT1. Bar = 100μm. Arrow indicates membrane staining. E) TPA treatment markedly increases 3-OMG uptake by WT but not 226A mutant transporters (n=10, error bars = SEM) in oocytes. F) Biotin pulldown of surface proteins demonstrates that endogenous GLUT1 increases its membrane (normalized against Na+/K+ ATPase) abundance after treatment with TPA in Rat2 cells; this is blocked by pre-treatment with a PKC inhibitor. HSP90 blot demonstrates the decrease in cytoplasmic protein concentration after biotin pulldown for membrane proteins. G) Quantitation of biotin pulldown experiments demonstrates significant increase in membrane GLUT1 induced by TPA (n=4, error bars = SEM). t-test, *p<0.05, **p<0.01, ***p<0.001. See also Figure S3.

Figure 3

Phosphorylation and localization of GLUT1 in primary endothelial cells. A) Endogenouse GLUT1 in human aortic endothelial cells (HAEC) is phosphorylated on S226 in response to TPA; the phosphorylation is blocked by a PKC inhibitor. B) HAEC increases 2DG uptake within 30 minutes of treatment by TPA. This increase is blocked by pretreatment with Gö-6983 (n=3, error bars = SD). C) Human umbilical vein endothelial cells (HUVEC) stained with DAPI (blue), total GLUT1 (green), and pGLUT1 S226 (red). Membrane staining of total GLUT1 (middle column) is apparent after TPA stimulation (bottom row). pGLUT1 (left column) shows a striking increase after TPA treatment with clear localization to cell membranes after TPA treatment. Arrows highlight regions of GLUT1 and pGLUT1 colocalization at membrane ruffles. Bar=25μm D) Total GLUT1 and pGLUT1 S226 staining in consecutive frozen sections from a patient biopsy sample (lymphatic malformation) showing partial overlap of GLUT1 and pGLUT1 staining. Both antibodies stain the perineurium as demonstrated by the rim of staining around the large peripheral nerve. E) HUVECs phosphorylate endogenous GLUT1 in response to GLUT1 in response to VEGF (100ng/ml) in a time dependent manner. F) Quantitation of pGLUT1 normalized to GAPDH showed significant differences in phosphorylation of GLUT1 in response to VEGF (n=5, error bars = SEM; ANOVA, p=0.004; Tukey’s, p

Figure 4

G1D mutations inhibit S226 phosphorylation and response to TPA. A) Sequence alignment of patient derived G1D mutations that highlight the loss of conserved residues of the optimal PKC consensus motifs. B) Patient derived G1D mutations surrounding the S226 motif decrease the ability of PKCβ1 to phosphorylate GST-GLUT1-Loop6 in vitro. Autoradiogram and Coommassie are representative of ≥3 independent experiments. C) Compared to WT GLUT1, K256E is stably expressed and shows comparable levels of phosphorylation after treatment with TPA. D) WT, R223P, R223Q, R223W, and 227insPPV are stably expressed in Rat2 fibroblasts. E) No significant difference between basal 2-DG uptake by WT compared to 226A, 223P, 223Q, 223W, 223Q, 227inPPV, or 256E mutant transporters (n≥3, error bars = SEM; Dunnett’s). F) 2-DG uptake by R223P, R223Q, R223W, and 227insPPV are impaired in their response to TPA [(n≥3, except K256E, n=2)]. G) Maximal TPA-induced increases in glucose uptake are impaired in naturally-occurring, pathogenic mutations surrounding S226.