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. 2012 Feb 14;109(7):2290-5.
doi: 10.1073/pnas.1119740109. Epub 2012 Jan 30.

Regulation of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) by reversible lysine acetylation

Chunfen Zhang  1 , Philip W Majerus, Monita P Wilson
Affiliations

Regulation of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) by reversible lysine acetylation

Chunfen Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

The enzyme inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) catalyzes the rate-limiting step in the formation of higher phosphorylated forms of inositol in mammalian cells. Because it sits at a key regulatory point in the inositol metabolic pathway, its activity is likely to be regulated. We have previously shown that ITPK1 is phosphorylated, a posttranslational modification used by cells to regulate enzyme activity. We show here that ITPK1 is modified by acetylation of internal lysine residues. The acetylation sites, as determined by mass spectrometry, were found to be lysines 340, 383, and 410, which are all located on the surface of this protein. Overexpression of the acetyltransferases CREB-binding protein or p300 resulted in the acetylation of ITPK1, whereas overexpression of mammalian silent information regulator 2 resulted in the deacetylation of ITPK1. Functionally, ITPK1 acetylation regulates its stability. CREB-binding protein dramatically decreased the half-life of ITPK1. We further found that ITPK1 acetylation down-regulated its enzyme activity. HEK293 cells stably expressing acetylated ITPK1 had reduced levels of the higher phosphorylated forms of inositol, compared with the levels seen in cells expressing unacetylated ITPK1. These results demonstrate that lysine acetylation alters both the stability as well as the activity of ITPK1 in cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CBP acetylates ITPK1 both in vitro and in vivo. (A) ITPK1 acetylation in transiently transfected HEK293 cells, treated with TSA and Nia. Immunoprecipitated ITPK1 was subjected to SDS-PAGE and Western blotting, probed with anti-acetylated lysine antibody (Upper) and anti-FLAG antibody (Lower). (B) Detection of ITPK1 in vivo acetylation by 3H-acetate labeling. Autoradiograph of 3H-acetate labeled samples (Upper). Western blotting with anti-FLAG antibody serves as a loading control (Lower). (C) Acetylation of ITPK1 in an in vitro acetylation assay. (D). Determination of the half-life for acetylation of ITPK, data were fitted as [CoASH] = 42.8 - 38.8 × e(-t/67.6).
Fig. 2.
Fig. 2.
SIRT1 deacetylates ITPK1 both in vitro and in vivo. (A) In vivo deacetylation of ITPK1 by SIRT1. HEK293 cells were cotransfected with FLAG–ITPK1 and CBP with or without varying amount of SIRT1. After immunoprecipitation (IP) with anti-FLAG M2 beads, the IP products were subjected to SDS-PAGE and Western blot analyses were done using antibody against either acetylated lysine (Upper) or FLAG (Lower). (B) In vitro deacetylation of ITPK1. Purified ITPK1, NAD+, and recombinant SIRT1 were incubated at 30 °C for the indicated times followed by SDS-PAGE and Western blotting with anti-acetylated lysine (Upper) and anti-FLAG antibody (Lower).
Fig. 3.
Fig. 3.
Identification of ITPK1 acetylation sites. (A) Mass-spectrometry identified the ITPK1 acetylation sites to be K340, K383, and K410. (B) Comparison of the acetylation level of wild-type ITPK1 with that of site-directed mutants of ITPK1. HEK293 cells were transfected with ITPK1 alone (lane 1) or cotransfected with ITPK1 (wild-type or mutants) and CBP. Western blot analysis of immunoprecipitated ITPK1 probed with anti-acetylated lysine antibody (Upper) and anti-FLAG antibody (Lower). (C) Schematic representation of the ITPK1 protein and its truncated mutants.
Fig. 4.
Fig. 4.
ITPK1 stability is decreased by acetylation. HeLa cells were transfected with ITPK1 alone (A) or cotransfected with ITPK1 and CBP (B). CHX (150 μg/mL) was added into the medium for the indicated times. The level of ITPK1 was detected by Western blotting with anti-FLAG antibody. Levels of β-actin are shown as a loading control. (C) Quantification of blots shown in A and B. Transfection with ITPK1 alone (○) and with ITPK1 and CBP (▪) is shown. (D) HeLa cells were transfected with the triple mutant K340,383,410R alone or cotransfected with K340,383,410R and CBP, with CHX added to the medium for the indicated times. Stability of ITPK1 is measured by Western blotting with β-actin as a loading control.
Fig. 5.
Fig. 5.
Lysine acetylation decreases ITPK1 enzymatic activity. (A) Schematic representation of the purification steps used to separate acetylated ITPK1 from unacetylated ITPK1. (B) Western blot analyses of separated proteins were performed with anti-FLAG antibody (Lower) and acetyl-lysine antibody (Upper). (C) Comparison of the enzymatic activity of acetylated ITPK1 to that of the unacetylated protein.
Fig. 6.
Fig. 6.
Quantification of soluble 3H-labeled inositol polyphosphates separated using anion exchange HPLC chromatography. HEK293 cells stably expressing ITPK1 were grown in medium containing 3H-inositol for 3 d, and induced with tetracycline for 2 d in the presence and absence of transfection with CBP. The soluble inositol-containing extracts were separated using a Whatman Partisphere 5 strong anion exchange HPLC column. Counts incorporated are normalized to protein level.
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