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* This work was supported in part by Grant DK30648 from the National Institutes of Health The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Recipient of a postdoctoral fellowship from the American Diabetes Association. § Recipient of a fellowship from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, CNPq, Brazil.
Phosphatidylinositol (PI) 3-kinases catalyze the formation of 3′-phosphoinositides, which appear to promote cellular responses to growth factors and such membrane trafficking events as insulin-stimulated translocation of intracellular glucose transporters. We report here the cloning of a novel PI 3-kinase, p170, from cDNA of insulin-sensitive mouse 3T3-L1 adipocytes. Mouse p170 utilizes PI and to a limited extent PI 4-P as substrates, in contrast to the PI-specific yeast VPS34 homolog PtdIns 3-kinase and the p110 PI 3-kinases, which phosphorylate PI, PI 4-P, and PI 4,5-P2. Mouse p170 is also distinct from PtdIns 3-kinase or the p110 PI 3-kinases in exhibiting a 10-fold lower sensitivity to wortmannin. Unique structural elements of p170 include C-terminal sequences strikingly similar to the phosphoinositide-binding C2 domain of protein kinase C isoforms, synaptotagmins, and other proteins. These features of mouse p170 are shared with a recently cloned Drosophila PI 3-kinase, DmPI3K_68D. Together, these proteins define a new class of PI 3-kinase likely influenced by cellular regulators distinct from those acting upon p110- or VPS34-like PI 3-kinases.
Multiple species of 3′-phosphorylated inositol lipids produced in reactions catalyzed by phosphatidylinositol (PI)
3-kinases are thought to be involved in cellular signaling and membrane trafficking pathways. A relatively large, constitutive pool of PI 3-phosphate is present in resting cells, while very low levels of PI 3,4-bisphosphate and PI 3,4,5-trisphosphate are rapidly increased in response to a number of external cellular stimuli (reviewed in Refs.
), which can utilize only PI as substrate. In contrast, a second category of PI 3-kinases, isoforms of the p110 PI 3-kinase, are capable of phosphorylating PI 4-phosphate and PI 4,5-bisphosphate at the 3′ position (
). These enzymes apparently contribute to the regulated pools of PI 3,4-P2 and PI 3,4,5-P3 stimulated by receptor or non-receptor tyrosine kinase activation (in the case of the isoforms p110 and p110β) or G-protein activation (in the case of p110γ). The existence of multiple PI 3-kinase isoforms suggests the influence of multiple signaling pathways on these enzymes and, possibly, divergent functions of the individual 3′-phosphoinositides.
A role for polyphosphoinositides as second messengers has been suggested by their activation of protein kinases such as certain protein kinase C species (
). An additional role for polyphosphoinositide products of p110 PI 3-kinases in regulated membrane trafficking is suggested by the requirement for yeast VPS34 PI 3-kinase activity in proper targeting of soluble hydrolases to the vacuole (
). The importance of PI 3-kinases in these and other pathways has been inferred from association and/or activation of the enzyme in receptor or downstream signaling complexes, but also from sensitivity to PI 3-kinase inhibitors such as wortmannin (reviewed in Ref.
) suggests that this compound is not a universally potent inhibitor of PI 3-kinases.
The significance of PI 3-kinase in insulin action suggested an examination of the particular enzymes involved. To this end we sought to identify PI 3-kinase isoforms that are present in insulin-responsive cells. We report here that mouse 3T3-L1 adipocytes contain, in addition to p110 isoforms and a VPS34 homolog, a novel PI 3-kinase, which we call p170. This protein is biochemically distinct from the p110 or VPS34 family in both substrate specificity and sensitivity to wortmannin. The unique structure of p170 includes a C-terminal region with striking sequence similarity to the phospholipid/inositol polyphosphate-binding C2 domain previously identified in many membrane-associated proteins. These observations suggest that p170 defines a novel category of PI 3-kinases potentially the target of distinct activators and capable of producing 3′-phosphoinositides essential to specific cellular functions.
RESULTS AND DISCUSSION
Using a strategy based on PCR amplification of conserved lipid kinase sequences, we identified four species of PI 3-kinase expressed in 3T3-L1 adipocytes.
J. V. Virbasius and M. P. Czech, unpublished results.
Two of these corresponded to two of the known isoforms of the p110 catalytic subunit of PI 3-kinase (p110 and p110β), while the third was found to be more closely related to the yeast VPS34 gene product (
). The fourth sequence, although highly similar to the catalytic region sequences of known PI 3-kinases, was clearly distinct and was characterized further. Screening of 600,000 plaques of an adipocyte cDNA library with a probe derived from this PCR product identified seven overlapping clones. The two that covered the maximum extent of the 5′ and 3′ end were fully sequenced and were found to encompass a total of 5076 base pairs. To verify that these clones included the full coding region of this species, RACE-PCR clones were obtained and sequenced. Four independent clones were sequenced. The longest of these extended the cDNA sequence by only 6 nucleotides, while each of the others ended within 5 nucleotides of the longest. We conclude that the sequence reported represents the full extent of its 5′ end.
The sequence of this cDNA includes an open reading frame of 4530 nucleotides, which is predicted to encode a protein of about Mr 170,000 (p170) including the sequence with high homology to the putative catalytic domain of PI 3-kinases (designated as region II, Fig. 1) originally obtained as a PCR product. The catalytic region of p170 is more similar to those of the p110 PI 3-kinases than to the VPS34 (Fig. 2A), sharing, for example, the additional charged residues in the putative substrate pocket (Fig. 2B, bracket) proposed to accommodate 4- and 4,5-phosphorylated phosphoinositide substrates (
). In addition, the p170 sequence displays significant similarity to the region just N-terminal to the probable catalytic domain, which is also conserved among a number of known PI 3-kinases (region I, Fig. 1, Fig. 2). Region I displays the most similarity to the analogous region of the various p110 isoforms.
Despite this similarity in regions I and II, p170 is distinguished from other PI 3-kinases in having additional sequences on the C-terminal side of the catalytic domain not found in other known mammalian PI 3-kinases. This region of p170 contains a striking similarity (Fig. 2C) to a domain denoted C2 originally identified in a number of protein kinase C isoforms (
). This C2 domain has been identified as a phospholipid/inositol polyphosphate binding motif, which in some cases is dependent on Ca2+ for lipid binding activity. This feature of p170 is shared with a recently identified novel PI 3-kinase in Drosophila called DmPI3K_68D (
), with which it also shares high levels of similarity in regions I and II (Fig. 2). Both mouse and Drosophila proteins, however, lack the conserved aspartate residues involved in Ca2+ coordination by the calcium-regulated synaptotagmin C2A domain (
The above considerations indicate that mouse p170 represents the first known mammalian form of a new class of PI 3-kinase molecules, which also includes DmPI3K_68D. In the N-terminal half of p170, however, there is little similarity to DmPI3K_68D, or to other PI 3-kinases. Of special note, there is no sequence resembling the potential SH3-binding proline-rich motif identified in DmPI3K_68D (
Previously identified PI 3-kinases can be divided into two groups based on substrate specificity. Three known isoforms of the p110 catalytic subunit are known to utilize PI as well as PI 4-phosphate and PI 4,5-bisphosphate as substrates. VPS34, however, utilizes only PI. In order to test this property of p170, the complete coding region of p170 was fused to GST-encoding sequences of a baculovirus expression vector, and the enzyme was expressed in Sf9 cells as a GST fusion protein. PI 3-kinase assays were carried out on the fusion protein bound to glutathione-agarose beads. To determine product and substrate specificity, we presented as substrate a crude brain lipid extract that contains a mixture of phosphoinositides and other phospholipids. The deacylated products of the reaction were analyzed by HPLC separation of the glycerophosphoinositol headgroups. The results confirm the identity of this p170 as a PI 3-kinase, since only 3-phosphorylated products are detectable (Fig. 3A). These products include a large peak of PI 3-P and a much smaller peak of PI 3,4-P2 (in a ratio that averaged about 100:2 based on peak heights). No PI 3,4,5-P3 is detectable under the conditions of this assay system. When assayed in parallel with the same substrate and reaction conditions, glutathione-agarose-bound GST-p110 produces PI 3-P as well as PI 3,4-P2 and PI 3,4,5-P3 in a ratio of about 100:15:15 (Fig. 3B), consistent with the well established specificity of this protein. The specific activity of GST-p170 toward PI was found to be approximately 3 nmol min−1 mg−1, similar to the reported value of 2 nmol min−1 mg−1 for both GST-p110 and GST-PI3K68_D (
). The substrate specificity of p170 is also consistent with that obtained with DmPI3K_68D, including the reported 20-fold lower specific activity of that protein relative to p110 with PI 4-P as a substrate (
). This confirms that p170 has a substrate specificity distinct from either p110 or PtdIns 3-kinase. However, the very low amount of PI 3,4-P2 produced under conditions where p110 produces much more significant quantities argues that p170 might be considered as physiologically more like PtdIns 3-kinase than like p110. An obvious caveat to this consideration is that substrate specificity in vitro may not accurately reflect the properties of the enzyme in vivo. In any case, even a small amount of a particular phosphoinositide in a critical cellular location might be physiologically significant.
PI 3-kinase activity has been shown to be stimulated in response to a variety of extracellular stimuli (
). The importance of PI 3-kinase activity in the pathways emanating from these stimuli has been further suggested by the parallel sensitivity of these enzymes and the resulting biological responses to the fungal product wortmannin. This compound inhibits all p110 isoforms and the mammalian VPS34-like PtdIns 3-kinase at low nanomolar concentrations (
). We tested the sensitivity of p170 to increasing amounts of wortmannin in assay reactions containing glutathione-agarose-bound GST-p170 using PI as a substrate (Fig. 4). Under these conditions p170 activity was inhibited only by relatively high concentrations of wortmannin (50% inhibition by approximately 40-50 nM wortmannin). Even at 100 nM wortmannin, a concentration widely used for in vitro or in vivo studies of PI 3-kinase activity, about 20% of the activity of p170 remained. By contrast, in parallel reactions the activity of GST-p110 PI 3-kinase was readily inhibited by low nanomolar concentrations of the same preparation of wortmannin (50% inhibition at 2 nM), in accord with previous reports with cellular or recombinant p110 (
). The results presented here indicate the existence of a novel PI 3-kinase class in mammalian cells.
The ability of p170 to produce PI 3-P suggests that it could contribute to the pool of PI 3-P in mammalian cells, perhaps functioning like VPS34 in the constitutive control of protein trafficking between membrane compartments. The much lesser ability of p170 to produce PI 3,4-P2 and its apparent inability to generate PI 3,4,5-P3 might call into question a role for this isoform in growth factor regulated processes known to stimulate polyphosphoinositide production. However, it cannot be assumed that the in vitro substrate preference coincides exactly with the activity of p170 on membrane lipids in intact cells. Factors such as substrate availability or presentation are likely not accurately reflected in vitro (discussed in Ref.
). Furthermore, even a small amount of PI 3,4-P2, if directed to critical cellular locations, could have physiological significance. Like other second messengers, the signal from a small amount of this phosphoinositide could be amplified in a cascade of subsequent signaling events. In any case our results argue for caution in the interpretation of results obtained by the use of inhibitors such as wortmannin. At low concentrations of this drug, p110 and VPS34-like PI 3-kinases may be inhibited, but others such as p170 may still be partially or fully functional. A recent report of a PI 4-kinase with wortmannin sensitivity similar to p170 PI 3-kinase (
) suggests that multiple enzymatic activities may be affected at such increased concentrations of wortmannin. Alternative methods will be required to dissect rigorously the distinct contributions to cell function made by the multiple PI 3-kinase species now known to be present in mammalian cells. The identification of p170 and isolation of its cDNA should facilitate experimental approaches to this problem.
We thank Dr. Bruce Spiegelman for the gift of the adipocyte cDNA library and Dr. Michael Waterfield for the bovine p110 cDNA.