Alterations in an inositol phosphate code through synergistic activation of a G protein and inositol phosphate kinases

Abstract

In mammals, many cellular stimuli evoke a response through G protein activation of phospholipase C, which results in the lipid-derived production of inositol 1,4,5-trisphosphate (IP(3)). Although it is well established that IP(3) is converted to numerous inositol phosphates (IPs) and pyrophosphates (PP-IPs) through the action of up to six classes of inositol phosphate kinases (IPKs), it is not clear that these metabolites are influenced by G protein signaling. Here we report that activation of Galpha(q) leads to robust stimulation of IP(3) to IP(8) metabolism. To expose flux through these pathways, genetic perturbation was used to alter IP homeostasis. Coupled expression of a constitutively active Galpha(q)QL and one or more IPK gene products synergistically generated dramatic changes in the patterns of intracellular IP messengers. Many distinct IP profiles were observed through the expression of different combinations of IPKs, including changes in previously unappreciated pools of IP(5) and IP(6), two molecules widely viewed as stable metabolites. Our data link the activation of a trimeric G protein to a plethora of metabolites downstream of IP(3) and provide a framework for suggesting that cells possess the machinery to produce an IPK-dependent IP code. We imply, but do not prove, that agonist-induced alterations in such a code would theoretically be capable of enhancing signaling complexity and specificity. The essential roles for IPKs in organism development and cellular adaptation are consistent with our hypothesis that such an IP code may be relevant to signaling pathways.

Fig. 1.

Six classes of evolutionarily conserved IPKs generate an IP code in mammalian cells: (i) IPMK (also known as IPK2 or ARG82), a multikinase capable of producing I(1,3,4,5,6)P₅ through the dual phosphorylation I(1,4,5)P₃ at the 3- and 6-kinase positions, as well as through a 5-kinase activity toward I(1,3,4,6)P₄; (ii) IPK1, an IP₅ 2-kinase responsible for the generation of IP₆; (iii) IP3K, a selective IP₃ 3-kinase that produces I(1,3,4,5)P₄ from I(1,4,5)P₃; (iv) ITPK1 (also known as IP56K), an I(1,3,4)P₃ 5- and 6-kinase capable of producing I(1,3,4,6)P₄ and I(1,3,4,5)P₄, and a reversible 1-kinase/phosphatase that regulates I(3,4,5,6)P₄ levels; (v) IP6K/IHPK, an IP₅ /IP₆ /IP₇ 5-kinase that generates diphosphoinositol phosphates (also known as PP-IPs), including 5-PP-IP₄ and 5-PP-IP₅; and (vi) VIP1, an IP₆ /IP₇ kinase that generates a unique species of PP-IP₅ (x-PP-IP₅) and (PP)₂-IP₄ (IP₈). In addition, the action of four IPs are relevant to this pathway: (i) INPP1, a lithium-inhibited I(1,4)P₂/I(1,3,4)P₃ 1-phosphatase; (ii) 5PT, an I(1,4,5)P₃/I(1,3,4,5)P₄ 5-phosphatase; (iii) PTEN, a phosphoinositide 3-phosphatase that also has been shown to dephosphorylate IP₅; and (iv) DIPP, which degrades PP-IPs. The lightning bolt represents GPCR and RTK pathways that activate PLC isoforms; the yellow sphere represents lithium, a pharmacological agent used to inhibit INPP1. Legend for colored arrows indicates the six kinase (solid) and four phosphatase (dotted) enzymes.

Fig. 2.

Enhanced detection of Gα_qQL-induced IP₃ production by the trapping of metabolites. Rat1 cells were plated at densities of 25,000 cells per milliliter and labeled metabolically for 72 h with myo-[³H]inositol as described in Methods. Twenty-four hours before harvest, cells were infected with control adenovirus (labeled Mock) or Gα_qQL adenovirus (labeled Gα_q). Lithium treatment was performed by adding LiCl to the cells at a final concentration of 10 mM 1 h before harvest. Soluble IPs were extracted by HCl and resolved by HPLC. Peaks were normalized to the total inositol counts present in the samples.

Fig. 3.

Coincident activation of IP₃ kinases along with Gα_qQL synergistically enhances inositol phosphate metabolism and distinct species of IP₄. HEK293T cells were plated at a density of 100,000 cells per milliliter and labeled metabolically for 72 h with myo-[³H]inositol. Twenty-four hours before harvest, cells were transfected by the addition of FuGENE6/DNA complexes directly into the labeling media. DNA complexes were prepared by mixing pcDNA3.1 plasmids expressing IPMK, IP3K, or ITPK1 with either pcDNA3.1 (Left) or pcDNA3.1-Gα_qQL (Right); 100 ng of each cDNA was used for the transfection, and empty pcDNA3.1 was used to bring the total DNA in each transfection to 500 ng per well. Soluble IPs were extracted from the cells by using HCl and resolved by HPLC.

Fig. 4.

Detection of a flux of IP₅ emanating from activated Gα_q through the coexpression of IP₅/IP₆ kinases. (A) Capture of a flux of IP₅ synthesis by IP₅ kinases. myo-[³H]inositol-labeled cells were transfected with plasmids expressing IP6K1, IPK1, or IP6K1 and IPK1 with either pcDNA3.1 (Left) or pcDNA3.1-Gα_qQL (Right). Soluble IPs were extracted from the cells by using HCl and resolved by HPLC. (B) Enzymatic analysis of IP6K generated IP pyrophosphates. HEK293T cells were metabolically labeled and transfected with Gα_qQL and IP6K1 (Left) or Gα_qQL, IP6K1, and IPK1 (Right). Cells were harvested in boiling hot 50 mM Tris, and tubes containing collected cell debris were further heated for 5 min in boiling water to minimize phosphatase activity against soluble IPs. Extracts were incubated in reaction buffer for 30 min at 37°C in the absence or presence of 1 μg of human DIPP. Reactions were halted by the addition of 0.5 M HCl, and samples were resolved by HPLC. Slight changes in the elution times of IPs in A and B are due to the use of different HPLC columns between the experiments.

Fig. 5.

Coexpression of activated Gα_q with four of the six classes of IP kinases stimulates production of IP₈. Gα_qQL and IPMK were coexpressed with IPK1, IPK1 + IP6K1, or IPK1 + IP6K + VIP1. Soluble IPs were extracted from the cells by using HCl and resolved by HPLC. Note the changes in the scale of the x and y axes from previous figures.