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Inositol 1,3,4-Trisphosphate Acts in Vivo as a Specific Regulator of Cellular Signaling by Inositol 3,4,5,6-Tetrakisphosphate

1999; Elsevier BV; Volume: 274; Issue: 27 Linguagem: Inglês

10.1074/jbc.274.27.18973

1083-351X

Xiaonian Yang, Marco T. Rudolf, Mark A. Carew, Masako Yoshida, Volkmar Nerreter, Andrew M. Riley, Sung-Kee Chung, Karol S. Bruzik, Barry V. L. Potter, Carsten Schultz, Stephen B. Shears,

Cellular transport and secretion

Ca2+-activated Cl− channels are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092–14097), a novel second messenger that is formed after stimulus-dependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P3 inhibited Ins(3,4,5,6)P4 1-kinase activity that was either (i) in lysates of AR4–2J pancreatoma cells or (ii) purified 22,500-fold (yield = 13%) from bovine aorta. Next, we incubated [3H]inositol-labeled AR4–2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P3 2.7-fold, while [3H]Ins(3,4,5,6)P4 levels increased 2-fold; there were no changes to levels of other 3H-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P4 are regulated by Ins(1,3,4)P3 in vivo, independently of Ins(1,3,4)P3 being metabolized to Ins(3,4,5,6)P4. In addition, we found that the Ins(1,3,4)P3 metabolites, namely Ins(1,3)P2 and Ins(3,4)P2, were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P3 itself (IC50 = 0.17 μm). This result shows that dephosphorylation of Ins(1,3,4)P3 in vivo is an efficient mechanism to "switch-off" the cellular regulation of Ins(3,4,5,6)P4 levels that comes from Ins(1,3,4)P3-mediated inhibition of the 1-kinase. We also found that Ins(1,3,6)P3 and Ins(1,4,6)P3 were poor inhibitors of the 1-kinase (IC50 = 17 and >30 μm, respectively). The non-physiological trisphosphates,d/l-Ins(1,2,4)P3, inhibited 1-kinase relatively potently (IC50 = 0.7 μm), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P3 acts in an important signaling cascade. Ca2+-activated Cl− channels are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092–14097), a novel second messenger that is formed after stimulus-dependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P3 inhibited Ins(3,4,5,6)P4 1-kinase activity that was either (i) in lysates of AR4–2J pancreatoma cells or (ii) purified 22,500-fold (yield = 13%) from bovine aorta. Next, we incubated [3H]inositol-labeled AR4–2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P3 2.7-fold, while [3H]Ins(3,4,5,6)P4 levels increased 2-fold; there were no changes to levels of other 3H-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P4 are regulated by Ins(1,3,4)P3 in vivo, independently of Ins(1,3,4)P3 being metabolized to Ins(3,4,5,6)P4. In addition, we found that the Ins(1,3,4)P3 metabolites, namely Ins(1,3)P2 and Ins(3,4)P2, were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P3 itself (IC50 = 0.17 μm). This result shows that dephosphorylation of Ins(1,3,4)P3 in vivo is an efficient mechanism to "switch-off" the cellular regulation of Ins(3,4,5,6)P4 levels that comes from Ins(1,3,4)P3-mediated inhibition of the 1-kinase. We also found that Ins(1,3,6)P3 and Ins(1,4,6)P3 were poor inhibitors of the 1-kinase (IC50 = 17 and >30 μm, respectively). The non-physiological trisphosphates,d/l-Ins(1,2,4)P3, inhibited 1-kinase relatively potently (IC50 = 0.7 μm), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P3 acts in an important signaling cascade. inositol polyphosphate, wheren is the number of phosphates (e.g. InsP3) polyethylene glycol phospholipase C high pressure liquid chromatography 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane calmodulin-dependent protein kinase 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate hexakis (acetoxymethyl) ester There is considerable interest in the idea that Ins(1,4,5)P31and Ins(1,3,4,5)P4 (Fig. 1) act in a co-ordinated manner as mediators of stimulus-dependent Ca2+mobilization (1Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3313) Google Scholar, 2Irvine R.F. Putney Jr., J.W. Advances in Second Messenger and P hosphoprotein Research. Raven Press, New York1992: 161-185Google Scholar). This has naturally led us to consider that the 5-phosphatases that degrade Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (3Hansen C.A. Johanson R.A. Williamson M.T. Williamson J.R. J. Biol. Chem. 1987; 262: 17319-17326Abstract Full Text PDF PubMed Google Scholar) are signaling "off-switches." This in turn has created the impression that the pathway by which these two inositol phosphates are dephosphorylated serves only as a conduit that replenishes the free inositol pool. In contrast, we have recently suggested that one of these downstream products, namely Ins(1,3,4)P3, should be viewed in an important cell-signaling context (4Tan Z. Bruzik K.S. Shears S.B. J. Biol. Chem. 1997; 272: 2285-2290Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). This new hypothesis comes from the observation that a rat hepatic Ins(3,4,5,6)P41-kinase was inhibited in vitro by Ins(1,3,4)P3(4Tan Z. Bruzik K.S. Shears S.B. J. Biol. Chem. 1997; 272: 2285-2290Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 5Craxton A. Erneux C. Shears S.B. J. Biol. Chem. 1994; 269: 4337-4342Abstract Full Text PDF PubMed Google Scholar). The reason that this effect of Ins(1,3,4)P3 upon Ins(3,4,5,6)P4 metabolism is of such interest is that Ins(3,4,5,6)P4 is an inhibitor of the conductance of the calcium-activated Cl− channels in the plasma membrane (6Ho M.W.Y. Shears S.B. Bruzik K.S. Duszyk M. French A.S. Am. J. Physiol. 1997; 272: 1160-1168Crossref PubMed Google Scholar, 7Vajanaphanich M. Schultz C. Rudolf M.T. Wasserman M. Enyedi P. Craxton A. Shears S.B. Tsien R.Y. Barrett K.E. Traynor-Kaplan A.E. Nature. 1994; 371: 711-714Crossref PubMed Scopus (178) Google Scholar, 8Xie W. Kaetzel M.A. Bruzik K.S. Dedman J.R. Shears S.B. Nelson D.J. J. Biol. Chem. 1996; 271: 14092-14097Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Ismailov I.I. Fuller C.M. Berdiev B.K. Shlyonsky V.G. Benos D.J. Barrett K.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10505-10509Crossref PubMed Scopus (94) Google Scholar). These ion channels make important contributions to salt and fluid secretion, and in addition they may participate in osmoregulation, pH balance, and smooth muscle excitability (10Nauntofte B. Am. J. Physiol. 1992; 263: G823-G837PubMed Google Scholar, 11Petersen O.H. J. Physiol. 1992; 448: 1-51Crossref PubMed Scopus (368) Google Scholar, 12Large W.A. Wang Q. Am. J. Physiol. 1996; 271: C435-C454Crossref PubMed Google Scholar, 13Barrett K.E. Am. J. Physiol. 1993; 265: C859-C868Crossref PubMed Google Scholar). The cellular accumulation of Ins(3,4,5,6)P4 is known to correlate well with receptor-dependent changes in PLC activity, but the molecular mechanisms that link these two events have not been fully elucidated (14Menniti F.S. Oliver K.G. Putney Jr., J.W. Shears S.B. Trends Biochem. Sci. 1993; 18: 53-56Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Our current hypothesis (15Menniti F.S. Oliver K.G. Nogimori K. Obie J.F. Shears S.B. Putney Jr., J.W. J. Biol. Chem. 1990; 265: 11167-11176Abstract Full Text PDF PubMed Google Scholar, 16Oliver K.G. Putney Jr., J.W. Obie J.F. Shears S.B. J. Biol. Chem. 1992; 267: 21528-21534Abstract Full Text PDF PubMed Google Scholar) is that cellular levels of Ins(3,4,5,6)P4 depend upon a dynamic balance between two competing enzyme activities acting in a closed substrate cycle: Ins(1,3,4,5,6)P5 1-phosphatase and Ins(3,4,5,6)P4 1-kinase (Fig. 1). The poise of this cycle is proposed to be regulated in such a manner that it can shift in favor of Ins(3,4,5,6)P4 accumulation whenever PLC is activated, perhaps through inhibition of the Ins(3,4,5,6)P4 1-kinase by Ins(1,3,4)P3 (Fig. 1). However, to date such inhibition has only been observed in studies with the purified rat hepatic kinase (4Tan Z. Bruzik K.S. Shears S.B. J. Biol. Chem. 1997; 272: 2285-2290Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 5Craxton A. Erneux C. Shears S.B. J. Biol. Chem. 1994; 269: 4337-4342Abstract Full Text PDF PubMed Google Scholar). No direct evidence has previously been published that indicates Ins(1,3,4)P3 can regulate Ins(3,4,5,6)P41-kinase activity in intact cells; it was a goal of the current study to explore this issue. In order to investigate if Ins(1,3,4)P3 can regulate Ins(3,4,5,6)P4 levels in intact cells, it was necessary to increase cellular levels of Ins(1,3,4)P3 specifically, under conditions where PLC activity was not activated. In this way, we could avoid the possibility of Ins(3,4,5,6)P4 metabolism also being regulated by the many additional signal transduction processes that are activated downstream of PLC. To this end, the development of cell-permeant, bioactivatable analogues of inositol phosphates (7Vajanaphanich M. Schultz C. Rudolf M.T. Wasserman M. Enyedi P. Craxton A. Shears S.B. Tsien R.Y. Barrett K.E. Traynor-Kaplan A.E. Nature. 1994; 371: 711-714Crossref PubMed Scopus (178) Google Scholar, 17Li W. Schultz C. Llopis J. Tsien R.Y. Tetrahedron. 1997; 53: 12017-12040Crossref Scopus (66) Google Scholar, 18Rudolf M.T. Li W. Wolfson N. Traynor-Kaplan A.E. Schultz C. J. Med. Chem. 1998; 41: 3635-3644Crossref PubMed Scopus (23) Google Scholar) has provided us with new opportunities to examine the functions of inositol polyphosphates in intact cells. The charge-masking groups that enable these derivatives to permeate into cells are hydrolyzed by intracellular esterases, releasing the native isomer (7Vajanaphanich M. Schultz C. Rudolf M.T. Wasserman M. Enyedi P. Craxton A. Shears S.B. Tsien R.Y. Barrett K.E. Traynor-Kaplan A.E. Nature. 1994; 371: 711-714Crossref PubMed Scopus (178) Google Scholar, 17Li W. Schultz C. Llopis J. Tsien R.Y. Tetrahedron. 1997; 53: 12017-12040Crossref Scopus (66) Google Scholar). In this study we used a new cell-permeant analogue, 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate hexakis(acetoxymethyl) ester (Bt3Ins(1,3,4)P3/AM; Ref. 19Rudolf M.T. Traynor-Kaplan A.E. Schultz C. Bioorg. Med. Chem. Lett. 1998; 8: 1857-1860Crossref PubMed Scopus (12) Google Scholar), to elevate the cellular concentration of Ins(1,3,4)P3 inside rat pancreatoma (AR4–2J) cells. This experimental approach was important for another reason. Rather than Ins(1,3,4)P3 regulating an enzyme of Ins(3,4,5,6)P4 metabolism (Fig.1), in principle, Ins(1,3,4)P3 could instead elevate Ins(3,4,5,6)P4 concentration simply by being metabolized to it (i.e. a mass action effect). For example, Ins(1,3,4)P3 can be converted to Ins(3,4,5,6)P4by the sequential actions of Ins(1,3,4)P3 6-kinase, Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5,6)P51-phosphatase (15Menniti F.S. Oliver K.G. Nogimori K. Obie J.F. Shears S.B. Putney Jr., J.W. J. Biol. Chem. 1990; 265: 11167-11176Abstract Full Text PDF PubMed Google Scholar, 16Oliver K.G. Putney Jr., J.W. Obie J.F. Shears S.B. J. Biol. Chem. 1992; 267: 21528-21534Abstract Full Text PDF PubMed Google Scholar, 20Shears S.B. J. Biol. Chem. 1989; 264: 19879-19886Abstract Full Text PDF PubMed Google Scholar). Others have proposed an alternative pathway for de novo Ins(3,4,5,6)P4 synthesis, which requires the sequential actions of Ins(1,3,4)P3 6-kinase, Ins(1,3,4,6)P4 1-phosphatase, and Ins(3,4,6)P35-kinase (21Stephens L.R. Hawkins P.T. Downes C.P. Biochem. J. 1989; 262: 727-737Crossref PubMed Scopus (28) Google Scholar, 22Stephens L.R. Berrie C.P. Irvine R.F. Biochem. J. 1990; 269: 65-72Crossref PubMed Scopus (13) Google Scholar, 23Stephens L.R. Downes C.P. Biochem. J. 1990; 265: 435-452Crossref PubMed Scopus (53) Google Scholar). We have now used AR4–2J cells to examine whether Ins(1,3,4)P3 alters Ins(3,4,5,6)P4 levels by mass action effects. Our strategy was based upon first prelabeling the metabolic pool of Ins(3,4,5,6)P4 to steady-state with [3H]inositol. These cells were then treated with non-radiolabeled Bt3Ins(1,3,4)P3/AM. We investigated if there was any significant metabolic flux from Ins(1,3,4)P3 to Ins(3,4,5,6)P4, which would have revealed itself by tending to decrease the amount of3H label in the [3H]Ins(3,4,5,6)P4 pool, due to a pulse-chase effect (15Menniti F.S. Oliver K.G. Nogimori K. Obie J.F. Shears S.B. Putney Jr., J.W. J. Biol. Chem. 1990; 265: 11167-11176Abstract Full Text PDF PubMed Google Scholar). Another feature of an effective signal transduction process relates to its specificity. If the biological effects of a signaling compound cannot be imitated by its products and precursors, this provides sensitivity in the signaling "on" and "off" switches. In the case of signaling by Ins(1,3,4)P3, the "on-switch" is dephosphorylation of Ins(1,3,4,5)P4 (3Hansen C.A. Johanson R.A. Williamson M.T. Williamson J.R. J. Biol. Chem. 1987; 262: 17319-17326Abstract Full Text PDF PubMed Google Scholar). This process is particularly sensitive, as Ins(1,3,4,5)P4 is a 290-fold weaker inhibitor of the 1-kinase than is Ins(1,3,4)P3 (4Tan Z. Bruzik K.S. Shears S.B. J. Biol. Chem. 1997; 272: 2285-2290Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). We have now turned our attention to considering how effective is the dephosphorylation of Ins(1,3,4)P3 as a signaling off-switch. In vivo, both 4- and 1-phosphatases actively degrade Ins(1,3,4)P3 to Ins(1,3)P2 and Ins(3,4)P2, respectively (24Bansal V.S. Inhorn R.C. Majerus P.W. J. Biol. Chem. 1987; 262: 9444-9447Abstract Full Text PDF PubMed Google Scholar, 25Inhorn R.C. Bansal V.S. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2170-2174Crossref PubMed Scopus (61) Google Scholar, 26Shears S.B. Kirk C.J. Michell R.H. Biochem. J. 1987; 248: 977-980Crossref PubMed Scopus (10) Google Scholar). We have therefore determined the potency with which these bisphosphate degradation products inhibit the 1-kinase. There was one further aspect to this study that is relevant to the development of Ins(3,4,5,6)P4 agonists and antagonists for pharmacological intervention in the signaling actions of Ins(3,4,5,6)P4 (18Rudolf M.T. Li W. Wolfson N. Traynor-Kaplan A.E. Schultz C. J. Med. Chem. 1998; 41: 3635-3644Crossref PubMed Scopus (23) Google Scholar, 27Schultz C. Roemer S. Stadler C. Rudolf M.T. Wolfson E. Traynor-Kaplan A.E. Gastroenterology. 1997; 112: A401Google Scholar). This goal is directed at diseases that might be treated by either up-regulating or down-regulating Ca2+-activated Cl− secretion (18Rudolf M.T. Li W. Wolfson N. Traynor-Kaplan A.E. Schultz C. J. Med. Chem. 1998; 41: 3635-3644Crossref PubMed Scopus (23) Google Scholar, 27Schultz C. Roemer S. Stadler C. Rudolf M.T. Wolfson E. Traynor-Kaplan A.E. Gastroenterology. 1997; 112: A401Google Scholar). A major challenge to pharmacological intervention at the effector site for Ins(3,4,5,6)P4 comes from the exquisite specificity with which it blocks Cl− channel conductance; Ins(1,3,4)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, and Ins(1,3,4,5,6)P5 are all ineffective (6Ho M.W.Y. Shears S.B. Bruzik K.S. Duszyk M. French A.S. Am. J. Physiol. 1997; 272: 1160-1168Crossref PubMed Google Scholar, 8Xie W. Kaetzel M.A. Bruzik K.S. Dedman J.R. Shears S.B. Nelson D.J. J. Biol. Chem. 1996; 271: 14092-14097Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Ismailov I.I. Fuller C.M. Berdiev B.K. Shlyonsky V.G. Benos D.J. Barrett K.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10505-10509Crossref PubMed Scopus (94) Google Scholar). Moreover, at least one, and possibly both of the OH groups on Ins(3,4,5,6)P4, are also critical determinants of substrate specificity (18Rudolf M.T. Li W. Wolfson N. Traynor-Kaplan A.E. Schultz C. J. Med. Chem. 1998; 41: 3635-3644Crossref PubMed Scopus (23) Google Scholar, 28Xie W. Solomons K.R.H. Freeman S. Kaetzel M.A. Bruzik K.S. Nelson D.J. Shears S.B. J. Physiol. 1998; 510: 661-673Crossref PubMed Scopus (49) Google Scholar). This tight specificity may make it difficult to rationally design a functional analogue of Ins(3,4,5,6)P4. A possible alternative therapeutic strategy might be to target Ins(3,4,5,6)P4 synthesis, rather than its site of action. To this end, we examined the impact on the 1-kinase of some analogues of Ins(1,3,4)P3 that contain a phosphate group in the 2-position. [3H]Inositol was purchased from American Radiolabeled Chemicals Inc. or NEN Life Science Products. [3H]Ins(1,3,4)P3 was prepared by dephosphorylating [3H]Ins(1,3,4,5)P4 (20 Ci/mmol, NEN Life Science Products) with recombinant Ins(1,3,4,5)P4 5-phosphatase, which was kindly provided by Dr. C. Erneux (29De Smedt F. Verjans B. Mailleux P. Erneux C. FEBS Lett. 1994; 347: 69-72Crossref PubMed Scopus (67) Google Scholar). Ins(1,4)P2 was purchased from Sigma.d/l-2,5,6-Tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester (d/l-Bt3Ins(1,3,4)P3/AM) was synthesized as described previously (19Rudolf M.T. Traynor-Kaplan A.E. Schultz C. Bioorg. Med. Chem. Lett. 1998; 8: 1857-1860Crossref PubMed Scopus (12) Google Scholar). In some experiments, we used enantiomerically pured-Bt3Ins(1,3,4)P3/AM. This compound was prepared from the enantiomerically pure precursor, 4-O-benzyl-1,2:5,6-di-O-cyclohexylidene-myo-inositol (30Rudolf M.T. Kaiser T. Guse A.H. Mayr G.W. Schultz C. Liebigs Ann./Recueil. 1997; 9: 1861-1869Crossref Scopus (17) Google Scholar). Alkylation of the hydroxy group with benzyl bromide in dimethyl formamide at 50 °C for 20 h and in the presence of an excess of sodium hydride and tetrabutyl ammonium iodide afforded 3,4-di-O-benzyl-1,2:5,6-di-O-cyclohexylidene-myo-inositol. Purification by preparative HPLC (92% MeOH, RP-18, 10 μm, 50 × 250 mm, 40 ml/min) gave 55% yield as an oil [α]D20 was +23°; c = 0.75, chloroform). The more labile ketal was removed by a 2-h treatment with acetyl chloride (5%) in a mixture of acetonitrile and methanol (4:5, v/v). The solution was neutralized with triethylamine and evaporated to dryness. The crude material was purified by preparative HPLC (90% MeOH, RP-18, 10 μm, 50 × 250 mm, 40 ml/min) to give 3,4-di-O-benzyl-1,2-O-cyclohexylidene-myo-inositol in 58% yield as a clear oil [α]D20was −11.1°; c = 0.72, chloroform). The latter was finally converted tod-Bt3Ins(1,3,4)P3/AM, as described previously for the racemic precursor (19Rudolf M.T. Traynor-Kaplan A.E. Schultz C. Bioorg. Med. Chem. Lett. 1998; 8: 1857-1860Crossref PubMed Scopus (12) Google Scholar). Analytical data ford-Bt3Ins(1,3,4)P3/AM were as follows: [α]D20 was −7.3° (c = 0.87, toluene). Direct chemical ionization high resolution mass spectroscopy [M-CH2OAc]–(C33H52O28P3) gave a calculated m/z of 989.1858; found m/z was 989.1868. 1H and 31P NMR data were in accordance with those of the racemic compound. Enantiomerically pured-Bt2Ins(1,4,5,6)P4/AM was synthesized as described previously (7Vajanaphanich M. Schultz C. Rudolf M.T. Wasserman M. Enyedi P. Craxton A. Shears S.B. Tsien R.Y. Barrett K.E. Traynor-Kaplan A.E. Nature. 1994; 371: 711-714Crossref PubMed Scopus (178) Google Scholar). All bioactivatable esters were dissolved in Me2SO/Pluronic (5%, v/v) as described previously (7Vajanaphanich M. Schultz C. Rudolf M.T. Wasserman M. Enyedi P. Craxton A. Shears S.B. Tsien R.Y. Barrett K.E. Traynor-Kaplan A.E. Nature. 1994; 371: 711-714Crossref PubMed Scopus (178) Google Scholar). Ins(1,3)P2, d/l-Ins(3,4)P2,d/l-Ins(1,2,4)P3, Ins(1,2,3)P3,d/l-Ins(1,2,4,6)P4, andd/l-Ins(1,2,3,4)P4 were prepared as described previously (31Chung K. Chang Y.T. Kwon Y.U. J. Carbohydr. Chem. 1998; 17: 369-384Crossref Scopus (13) Google Scholar, 32Chung S.K. Chang Y.T. J. Chem. Soc. Chem. Commun. 1995; : 11-12Crossref Google Scholar, 33Chung S.K. Chang Y.T. Sohn K.H. J. Chem. Soc. Chem. Commun. 1996; : 163-164Crossref Scopus (29) Google Scholar). Ins(1,3,6)P3 and Ins(1,4,6)P3 were prepared as described previously (34Riley A.M. Payne R. Potter B.V.L. J. Med. Chem. 1994; 37: 3918-3927Crossref PubMed Scopus (26) Google Scholar,35Mills S.J. Potter B.V.L. J. Org. Chem. 1996; 61: 8980-8987Crossref PubMed Scopus (28) Google Scholar). Sources of other inositol phosphates are given elsewhere (4Tan Z. Bruzik K.S. Shears S.B. J. Biol. Chem. 1997; 272: 2285-2290Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar,8Xie W. Kaetzel M.A. Bruzik K.S. Dedman J.R. Shears S.B. Nelson D.J. J. Biol. Chem. 1996; 271: 14092-14097Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Bombesin, bovine serum albumin, phosphocreatine, phosphocreatine kinase, heparin agarose resin (type II and IIIs), and protease inhibitors were purchased from Sigma. The calmodulin-dependent protein kinase (CaM KII) was obtained from New England Biolabs. Protein kinases A and C, and their assay kits (SpinZyme Format), were the products of Pierce. The UNO Q12 anion exchange column was acquired from Bio-Rad Laboratories. Polyethylene glycol 4000 was purchased from Fluka. Frozen bovine aorta were purchased from Pel-Freez Biological. The 1-kinase activity was assayed as described before (4Tan Z. Bruzik K.S. Shears S.B. J. Biol. Chem. 1997; 272: 2285-2290Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, 10–20 μl of enzyme was incubated at 37 °C in a final volume of 100 μl containing about 4000 dpm [3H]Ins(3,4,5,6)P4, which was adjusted to a concentration of 0.25 μm with non-radioactive substrate, 20 mm HEPES (pH 7.2), 6 mm MgSO4, 0.4 mg/ml saponin, 100 mmKCl, 0.3 mg/ml bovine serum albumin, 2 μmInsP6, 5 mm ATP, 10 mmphosphocreatine, and 2.5 Sigma units of phosphocreatine kinase. After 30 min., the reaction was stopped by quenching with 1 ml of ice-cold medium containing 1 mg/ml InsP6, 0.2 m ammonium formate, and 0.1 m formic acid. The quenched reactions were diluted to 10 ml with deionized water, and chromatographed on Bio-Rad gravity-fed columns using AG 1-X8 ion exchange resin. For some assays, the 1-kinase was preincubated at 30 °C for 10 min with (a) 125 units of the catalytic subunit of protein kinase A, (b) 0.2 unit of protein kinase C, (c) 600 units of calmodulin, or (d) 500 units of CaM KII, preactivated with calmodulin/Ca2+ (New England Biolabs). The protein kinases used in these experiments were all shown to be active in control experiments (assay kits for protein kinases A and C were supplied by Pierce; the CaM KII was checked using a kit purchased from Upstate Biochemicals). The 1-kinase was also used as a diagnostic tool to verify the nature of HPLC-purified [3H]Ins(3,4,5,6)P4. In these incubations, 45 μl of purified 1-kinase was added to 225 μl of medium containing 67 mm HEPES (pH 8.0 with KOH), 0.7 mm EDTA, 8.7 mm MgSO4, 6.7 mm ATP, 13.3 mm phosphocreatine, 1.33 μm InsP6, and 6 Sigma units of phosphocreatine kinase. Then, 30 μl of the appropriate HPLC fraction was added (which brought the final pH to approximately 6.5). Reactions (at 37 °C) were allowed to proceed to completion (over a 3-h period), and then the amount of [3H]InsP5formed was determined using gravity-fed ion-exchange columns, as described above. Frozen bovine aortas were thawed on ice, the attached fat was removed, and then the aorta were pulverized in a meat grinder. In a typical preparation, 300–350 g of ground aortas were homogenized in two volumes of 50 mm bis-Tris (pH 7.0), 1 mm EGTA, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mmphenylmethylsulfonyl fluoride in a tissue blender. The homogenate was filtered through four layers of cheesecloth, and a 10–30% (w/v) polyethylene glycol 4000 precipitate was prepared. The resultant pellet was resuspended in 100 ml of Buffer A containing 50 mm bis-Tris (pH 7.0), 1 mm EGTA, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A. The suspension was filtered and loaded at a flow rate of 1 ml/min onto a heparin-agarose type II column (3.2 × 24 cm). After washing with 300 ml of Buffer A at flow rate of 1.5 ml/min, the bound protein was eluted with a linear gradient of 0–30 mm of sodium pyrophosphate in Buffer A. The peak fractions of enzyme activity eluted from the heparin column were pooled, then frozen and stored at −70 °C. Either two or three preparations were subsequently thawed and combined, dialyzed against 2 liters of 25 mm bis-Tris (pH 7.0) at 4 °C for 3 h, and loaded onto a UNO Q12 anion exchange column (1.5 × 6.8 cm), which was pre-equilibrated with 100 ml of Buffer A. A constant flow rate of 0.5 ml/min was maintained throughout the chromatography. After washing with 60 ml of Buffer A, the bound protein was eluted with a linear gradient of Buffer A plus 0–300 mm NaCl, followed by 60 ml of Buffer A plus1 m NaCl. Peak fractions of enzyme activity eluted from the UNO Q12 column were pooled, dialyzed against 2 liters of 25 mm bis-Tris (pH 7.0) at 4 °C for 3 h, and loaded on to heparin-agarose type IIIs (1.1 × 13.5 cm), which was pre-equilibrated with 50 ml of Buffer A. A constant flow rate of 0.5 ml/min was maintained throughout. After washing with 60 ml of Buffer A, the bound protein was eluted with a linear gradient of 0–300 mm NaCl in Buffer A, followed by 60 ml of 1 m NaCl in Buffer A. The protein concentration of the 1-kinase preparation was determined using Bio-Rad Protein Assay Dye Reagent with bovine serum albumin as standard. Final enzyme preparations were stored in 10% glycerol plus 1 mg/ml bovine serum albumin at −70 °C. A 1-ml aliquot of a resuspension of a 10–30% PEG precipitation was loaded at a flow rate of 0.25 ml/min to Sephacryl S100 column (2.0 × 86 cm), which was pre-equilibrated with 600 ml of bis-Tris buffer containing 50 mm bis-Tris (pH 7.0), 1 mm EGTA, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 100 mm NaCl. The protein was then chromatographed using the same buffer at a constant 0.25 ml/min flow rate. Fractions (5ml) were collected and assayed for enzyme activity. The column was calibrated under the exactly same conditions using bovine serum albumin, chicken ovalbumin, equine myoglobin, and vitamin B-12. The AR4–2J pancreatoma cells were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 10% fetal bovine serum, 2 mm glutamine, 500 units/ml penicillin, and 500 μg/ml streptomycin, with 10% conditioned medium, and harvested by brief trypsinization. Either 2.0 × 105 or 1.2 × 106 cells were seeded in 24-well or 6-well tissue culture plates, respectively. Cells were labeled with 75–150 μCi/ml [3H]myo-inositol for 4 days (medium was replaced on the 3rd day) in 700 μl (for 24-well plates) or 3 ml (for 6-well plates) of the above culture medium. After completion of the labeling protocol, the culture medium was aspirated and the cells were washed twice with Krebs/Ringer/HEPES solution (15Menniti F.S. Oliver K.G. Nogimori K. Obie J.F. Shears S.B. Putney Jr., J.W. J. Biol. Chem. 1990; 265: 11167-11176Abstract Full Text PDF PubMed Google Scholar). Cells were then incubated in 300 μl (for 24-well) or 1 ml (for 6-well) of Krebs/Ringer/HEPES solution for 2 h. Then 20 mm LiCl was added, and 20 min later cells were treated for the indicated time with (i) a cell-permeant inositol phosphate, (ii) vehicle, or (iii) bombesin. Cells were quenched and neutralized, and the inositol phosphates were separated by HPLC as described elsewhere (36Shears S.B. Shears S.B. Signalling by Inositides: A Practical Approach. Oxford University Press, Oxford, United Kingdom1997: 33-52Google Scholar). Radioactivity was either counted on-line, using a Radiomatic Flo-1, or recovered in 1-ml fractions. The levels of 3H-labeled inositol phosphates were normalized as a ratio to cellular levels of [3H]InsP6; the latter were unaffected by any of the experimental protocols performed in this study. For some experiments, after the AR4-2J cells were harvested, cells were collected by centrifugation in serum-containing culture medium. The pellet was washed in HEPES-buffered saline, and then a lysate was prepared by resuspending the packed cells in an equal volume of ice-cold lysis buffer comprising: 50 mm KCl, 50 mm HEPES (pH 7.2), 1 mm EDTA, 5 mmATP, 4 mm CHAPS, 0.4 mm phenylmethylsulfonyl fluoride, 40 μm E-64, 10 μm leupeptin, 3 μm pepstatin. Following the HPLC fractionation of extracts of [3H]inositol-labeled cells (see above), 1-ml fractions were saved, from which 25-μl aliquots were counted for radioactivity so as to identify the Ins(1,3,4)P3 peak (because of the low levels of endogenous [3H]Ins(1,3,4)P3, samples were "spiked" with 4000 dpm [3H]Ins(1,3,4)P3 (20 Ci/mmol) before they were applied to the HPLC column). The Ins(1,3,4)P3 peak was then desalted (37Maslanski J.A. Busa W.B. Irvine R.F. Methods in Inositide Research. Raven Press, New York1990: 109-122Google Scholar) and resuspended in 60 μl Ins(1,3,4)P36-kinase assay buffer: 50 mm KCl, 50 mm HEPES, pH 7.2, 10 mm phosphocreatine, 6 mm ATP, 8 mm MgSO4, 25 Sigma units/ml phosphocreatine kinase, 0.5 mg/ml bovine serum albumin. Recovery of [3H]Ins(1,3,4)P3 from the cell extract was typically 70–75%. Each Ins(1,3,4)P3 sample was then divided into two equal portions, named A and B. The Ins(1,3,4)P3 was depleted from portion B by its incubation for 60 min at 37 °C with 0.1 μg of recombinant Ins(