Figure 3. GKRP acetylation is critical for binding and retaining GK in the nucleus.

(A,B) Interaction between GKRP and human glucokinase (hGK). HeLa cells transfected with expression vectors for GKRP and hGK were lysed, protein immunoprecipitated with anti-Myc or anti-V5 antibodies, and immunoblotted by anti-His or anti-Myc antibodies. (C) Effect of K5 mutations on GKRP interaction with hGK. HeLa cells cotransfected with hGK and wild type or the indicated GKRP mutants were lysed, protein precipitated with an anti-Myc antibody, and blotted with an anti-His antibody. β-actin expression was used as internal control. (D) Immunofluorescence micrographs showing the subcellular locations of GKRP and hGK. HeLa cells transfected with Myc-tagged GKRP and V5/His-tagged GK were maintained in 5.5 mM or 25 mM glucose for 4 hr in the absence (−) or presence (+) of a mixture of the HDACIs NAM (5 mM) and TSA (1 μM). Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.