p110delta , a Novel Phosphatidylinositol 3-Kinase Catalytic Subunit That Associates with p85 and Is Expressed Predominantly in Leukocytes*

(Received for publication, January 23, 1997, and in revised form, May 20, 1997)

David Chantry Dagger , Anne Vojtek §, Adam Kashishian Dagger , Douglas A. Holtzman Dagger par , Christi Wood Dagger , Patrick W. Gray Dagger , Jonathan A. Cooper and Merl F. Hoekstra Dagger **

From Dagger  ICOS Corporation, Bothell, Washington 98021, § Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109,  Fred Hutchinson Cancer Research Center, Seattle, Washington 98104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We have identified a novel p110 isoform of phosphatidylinositol 3-kinase from human leukocytes that we have termed p110delta . In addition, we have independently isolated p110delta from a mouse embryo library on the basis of its ability to interact with Ha-RasV12 in the yeast two-hybrid system. This unique isoform contains all of the conserved structural features characteristic of the p110 family. Recombinant p110delta phosphorylates phosphatidylinositol and coimmunoprecipitates with p85. However, in contrast to previously described p110 subunits, p110delta is expressed in a tissue-restricted fashion; it is expressed at high levels in lymphocytes and lymphoid tissues and may therefore play a role in phosphatidylinositol 3-kinase-mediated signaling in the immune system.


INTRODUCTION

Phosphatidylinositol (PI)1 3-kinase was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylates PI and its phosphorylated derivatives at the 3'-hydroxyl of the inositol ring (1). The purification and subsequent molecular cloning of PI 3-kinase revealed that it is a heterodimer consisting of p85 and p110 subunits (2-5).

The p85 subunit acts to localize PI 3-kinase activity to the plasma membrane by virtue of the interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate local sequence context) in target proteins (6). Two isoforms of p85 have been identified: p85alpha , which is ubiquitously expressed, and p85beta , which is primarily found in brain and lymphoid tissues (7). The p110 subunit contains the catalytic domain of PI 3-kinase, and three isoforms of p110 have thus far been reported (alpha , beta , and gamma ) (3, 8, 9). The identification of p110gamma revealed additional complexity within this family of enzymes. p110gamma is most closely related to p110alpha and beta  (45-48% identity in the catalytic domain) but does not make use of p85 as a targeting subunit. p110gamma contains an additional domain termed a pleckstrin homology domain near the amino terminus. The pleckstrin homology domain allows interaction with the beta gamma subunits of heterotrimeric G proteins that appears to regulate its activity and subcellular localization (9).

Additional members of this growing gene family include more distantly related lipid and protein kinases such as Vps34, TOR1, and TOR2 of Saccharomyces cerevisiae (and their mammalian homologues such as FRAP and mTOR), the human ataxia telangiectasia gene product, and the catalytic subunit of DNA-dependent protein kinase (10).

The levels of phosphatidylinositol-3,4,5-triphosphate, the primary product of PI 3-kinase activation, are elevated upon treatment of cells with a wide variety of agonists (11). This observation has implicated PI 3-kinase activation in a diverse range of cellular responses including cell growth, differentiation, and apoptosis (1, 12, 13). The downstream targets of the phosphorylated lipids generated following PI 3-kinase activation have not been well characterized. However some isoforms of protein kinase C are directly activated by phosphatidylinositol-3,4,5-triphosphate in vitro. The protein kinase C-related protein kinase AKT has also been shown to be activated by PI 3-kinase, although the mechanism of this has yet to be determined (14).

PI 3-kinase also appears to be involved in a number of aspects of leukocyte activation. PI 3-kinase physically associates with the cytoplasmic domain of CD28, which is an important co-stimulatory molecule for the activation of T cells in response to antigen (15, 16). Activation of T cells through CD28 lowers the threshold for activation by antigen and increases the magnitude and duration of the proliferative response. These effects are linked to increases in the transcription of a number of genes including the T cell growth factor interleukin 2 (17). Mutation of CD28 such that it can no longer interact with PI 3-kinase leads to a failure to initiate interleukin-2 production, suggesting a critical role for PI 3-kinase in T cell activation (15). Based on studies using the PI 3-kinase inhibitor wortmannin, there is evidence that PI 3-kinase(s) is also required for some aspects of leukocyte signaling through G protein-coupled receptors (18).

We report here the cloning of novel human and murine p110 isoforms (p110delta ) with a highly restricted pattern of expression. p110delta is expressed predominantly in leukocytes and may therefore play a role in PI 3-kinase-mediated signaling in the immune system.


MATERIALS AND METHODS

Identification of Human p110delta by PCR

Degenerate oligonucleotide primers were designed for use in the PCR reaction based on sequences conserved in the catalytic domain of known PI 3-kinases. The sense primer was 5'-GCAGACGGATCCGGIGAYGAYHKIAGRCARGA-3' encoding the sequence GDDLRQD, and the antisense primer was 5'-GCAGACGAATTCRWRICCRAARTCIRYRTG-3' (where I is inosine, R is A or G, Y is C or T, H is A, C, or T, W is A or T, and K is G or T) encoding the amino acid sequence HIDFGH. BamHI and EcoRI restriction sites are underlined. PCR reactions consisted of 100 ng of cDNA template (from human peripheral blood mononuclear cells (PBMC) activated for 18 h with 10 ng/ml phorbol myristate acetate and 250 ng/ml calcium ionophore (Sigma)), 10 µg/ml oligonucleotide primers, 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 200 mM dNTPs, and 1 unit of Taq polymerase in a final volume of 100 µl. Reactions were performed using denaturation for 1 min at 94 °C, annealing at 60 °C for 2 min, and 3 cycles of extension for 1 min at 72 °C. The procedure was then repeated using a 56 °C annealing temperature for 3 cycles, 52 °C annealing temperature for 3 cycles, and 50 °C annealing temperature for 30 cycles. Amplified products were gel-purified, digested with BamHI and EcoRI, and subcloned into the vector pBluescript SKII+ (Stratagene) for sequencing. All DNA for sequencing was prepared using the Wizard Miniprep DNA purification system (Promega, Madison, WI). Sequencing was performed on the Applied Biosystems model 373 automated sequencer. Data bank searches were performed using the BLAST program, and protein and DNA alignments were made using the Geneworks program (Intelligenetics Inc., Mountain View, CA). One clone contained a 399-base pair insert that encoded a 133-amino acid open reading frame showing ~80% identity with p110beta .

Identification of a Full-length cDNA for Human p110delta

To identify a full-length cDNA (which we subsequently termed p110delta ), specific oligonucleotide primers were designed based on the sequence of the PCR product. The forward primer was 5'-CATGCTGACCCTGCAGATGAT-3', and the reverse primer was 5'-AACAGCTGCCCACTCTCTCGG-3'. These were used to screen the aforementioned human PBMC cDNA library. Successive rounds of PCR were first performed on pools of 100,000 clones and subsequently on smaller pools until a single clone (termed PBMC 249) was isolated by colony hybridization using the PCR product (labeled by random priming) as a probe. This cDNA was not full-length. Therefore, to identify longer cDNA clones, the same approach was used to screen a cDNA library from human macrophages (19). This led to the isolation of an additional cDNA (M928) that extended the cDNA sequence by 1302 base pairs at the 5' end. The remaining 5' end of the cDNA was obtained by 5' RACE PCR (CLONTECH, Palo Alto, CA.) Two antisense cDNA-specific oligonucleotide primers were designed at the 5' end of cDNA M928 for RACE PCR reactions. The primary RACE primer was 5'-GGGCCACATGTAGAGGCAGCGTTCCC-3'. The nested RACE primer was 5'-GGCCCAGGCAATGGGGCAGTCCGCC-3'. Marathon RACE reactions were set up using a human leukocyte Marathon-ready cDNA template and the Advantage core PCR reaction kit (CLONTECH) following manufacturer protocol. Touchdown PCR cycling conditions were modified to improve the specificity of the Marathon RACE PCR primary reaction as follows: denaturation at 94 °C for 2 min followed by 5 cycles of 94 °C for 30 s, annealing and extension at 72 °C for 3 min, 5 cycles of 94 °C for 30 s, annealing and extension at 70 °C for 3 min, and 25 cycles of 94 °C for 30 s, annealing and extension at 68 °C for 3 min.

The 5' RACE PCR products were gel-purified and subcloned into the TA vector PCRII (Invitrogen, San Diego, CA) according to manufacturer instructions. Three independent clones were sequenced.

Assembly of Epitope-tagged Human p110delta for Expression in Mammalian Cells

A full-length cDNA for p110delta was assembled from clones 249, 928, and the 5' RACE PCR products. The 5' RACE product ODH18 was used as a template in the PCR using the primers ODH5' FLAG, 5'-AGTTACGGATCCGGCACCATG(GACTACAAGGACGACGATGACAAG)CCCCCTGGGGTGGACTGCCC-3' and ODH3' Paste 5'-CCACATGTAGAGGCAGCGTTCC-3'. The 5' primer includes a BamHI site (underlined) and encodes the peptide sequence DYKDDDDK (shown in parentheses), which is recognized by the M2 anti-FLAG monoclonal antibody (Kodak Scientific Imaging Systems). The resulting PCR product was digested with BamHI and AflII and was ligated along with an AflII/PvuI fragment derived from the clone M928 and a PvuII/XbaI fragment derived from PBMC clone 249 into the BamHI/XbaI sites of the mammalian expression vector pcDNA3 (Invitrogen). The resulting plasmid, (pcDNA3:FLAG/p110delta ), uses the cytomegalovirus promoter to drive the expression of full-length p110delta preceded by an initiating methionine residue and the FLAG epitope.

Identification of Mouse p110delta by Yeast Two-hybrid Screen and cDNA Cloning

cDNA encoding a fragment (amino acids 141-291) of mouse p110delta termed Rip36 (Ras-interacting protein) was identified by screening a mouse embryo library using Ha-RasV12 as bait in the yeast two-hybrid system. The yeast two-hybrid system and the plasmids have been described (20). The 5' end of the cDNA was obtained by RACE PCR using the reaction conditions described in Vojtek et al. (21) and the following specific oligonucleotides: 1) for the reverse transcription reaction, 5'-CGCGGATCCTGCTGACACGCAATAAGCCG-3'; 2) cDNA-specific primer 1, 5'-TAGGCACCTGCAGATGTACTG-3'; and 3) cDNA-specific primer 2, 5'-CGCGGATCCTGCTGACACGCAATAAGCCG-3'. The template for the reverse transcription reaction was spleen RNA from adult mouse, prepared as described (21). Sequence analysis of cDNAs present after the RT-PCR reaction identified 558 nucleotides of p110delta coding sequence, corresponding to amino acids 1-186 and 36 nucleotides of 5'-untranslated region. An in-frame stop is present 12 nucleotides upstream of the predicted initiating ATG.

The 3' end of murine p110delta was obtained by RACE PCR using the Expand PCR system (Boehringer Mannheim) according to manufacturer instructions. The cDNA-specific oligonucleotides used for amplification are 5'-GCCAGTTTTGTGAAGAGGCTG-3' and 5'-AGCGGATCCCAGTACATCTGCAGGTGCCTAC-3'. A product of 3.6 kilobases was subcloned, restriction mapped, and sequenced. This cDNA contains 2,415 nucleotides of p110delta coding sequence followed by 3'-untranslated region.

A full-length cDNA for the mouse p110delta was obtained by reverse transcription of spleen RNA from adult mice followed by 35 cycles of PCR using the Expand system (Boehringer Mannheim) according to manufacturer instructions and the following two cDNA-specific oligonucleotides: 5'-GGAAGATCTTGGCGATGCCCCCTGGGGTGGACTGC-3' and 5'-GGAAGATCTGCGGCCGCCTACTGTCGGTTATCCTTG-3'. The latter oligonucleotide was also used in the reverse transcription reaction. The reverse transcription reaction contained the following components/10 µl reaction: 2 µg of total spleen RNA from adult mice, 4 pmol of the gene-specific primer, 1 µl of 10 mM dNTPs (U. S. Biochemical Corp.), 1 unit of RNasin (Promega), 200 units of Superscript reverse transcriptase (Life Technologies, Inc.), 2 µl of 5 × RT buffer supplied by the manufacturer (Life Technologies), 5 mM dithiothreitol. The RT reaction was incubated at 44 °C for 2 h. Five µl of the RT reaction was amplified by PCR in a reaction volume of 50 µl using the Expand system (Boehringer Mannheim). The reaction conditions were denaturation at 94 °C for 2 min followed by 35 cycles of denaturation at 94 °C for 20 s, annealing at 57 °C for 30 s, and extension at 68 °C for 3.5 min. The cDNA was subcloned directly to the pT7 blue T vector (Novagen) and subsequently transferred to the mammalian expression vector pEBG, which drives the expression of a glutathione S-transferase fusion protein, including the complete coding sequence of mouse p110delta (22).

Northern Blot Analysis

Radiolabeled [32P]cDNA probes were prepared by PCR using 10 ng of plasmid DNA template encoding p110delta . The forward primer was 5'-CTGCCATGTTGCTCTTGTTGA-3', and the reverse primer was 5'-GAGTTCGACATCAACATC-3'. PCR reactions contained 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 200 µM dATP, dTTP, dGTP, and 1 µM dCTP, 50 µCi of [alpha -32P]dCTP (DuPont NEN), 10 µg/ml cDNA-specific primers, and 2 units of Taq DNA polymerase. Reactions were heated for 4 min at 94 °C followed by 15 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 2 min at 72 °C. Unincorporated nucleotides were removed by passing the reaction over a Sephadex G-50 column (Boehringer Mannheim). A multiple tissue northern blot (CLONTECH) was probed and washed under stringent conditions according to manufacturer recommendations. The autoradiograph was exposed for 1-4 days at -80 °C with intensifying screens.

Expression of p110delta in Mammalian Cells and PI 3-Kinase Assay

The epitope-tagged p110delta was transfected into COS cells using DEAE dextran (23). Three days after transfection, the cells were serum-starved overnight in Dulbecco's modified Eagle's medium plus 0.1% fetal bovine serum. The plates were rinsed once with calcium- and magnesium-free phosphate-buffered saline and lysed in 3 ml of buffer R per confluent 150-mm plate (buffer R is 1% Triton X-100, 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 0.5% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, 1% aprotinin). p110delta was immunoprecipitated using the monoclonal antibody anti-FLAG M2 (Kodak Scientific Imaging) according to manufacturer recommendations. The immunoprecipitates were washed three times with buffer R and twice with PAN buffer (100 mM NaCl, 10 mM PIPES, 20 µg/ml aprotinin) and resuspended in PAN buffer. One-tenth of the immunoprecipitates were incubated for 15 min at 30 °C in a total volume of 10 µl containing 0.2 mg/ml phosphatidylinositol (Sigma), 20 mM HEPES (pH 7.4), 5 mM MnCl2, 0.45 mM EGTA, 10 µCi of [gamma -32P]ATP, 10 µM ATP. The reactions were terminated by the addition of 100 µl of 1 M HCl. The phospholipids were extracted once with 200 µl of chloroform/methanol (1:1 v/v) and once with 80 µl of HCl/methanol (1:1), lyophilized to dryness, resuspended in 10 µl of chloroform/methanol, and spotted onto a 1% potassium oxalate-impregnated silica 60 thin layer chromatography plate (V. W. R. Scientific). The phospholipids were resolved by ascending chromatography in chloroform, methanol, 4 M NH4OH (9:7:2) and visualized by autoradiography. Crude phospholipid standards (Sigma) were run in parallel with the radiolabeled samples and visualized by exposing the plate to iodine vapor.


RESULTS AND DISCUSSION

Using a strategy based on amplification of conserved PI 3-kinase sequences, we have identified a novel human member of this family that we have termed p110delta . A combination of cDNA library screening and 5' RACE PCR has led to the identification of cDNAs encompassing the complete coding region of p110delta . The deduced amino acid sequence of p110delta is shown in Fig. 1.


Fig. 1. Deduced amino sequence of human (Hu) and murine (Mu) p110delta . Identical residues are shown by dots, the Ras regulatory domain is underlined, and the start of the carboxyl-terminal catalytic domain is shown by a down-arrow .
[View Larger Version of this Image (48K GIF file)]

In an independent search for mouse Ha-RasV12-interacting proteins (Rips) using the yeast two-hybrid system, we identified two clones related in sequence to p110beta : Rip31 and Rip36. Using a PCR-based strategy and gene-specific oligonucleotide primers derived from the Rip36 sequence, a full-length cDNA was isolated (see "Materials and Methods"). Sequence analysis suggests that this clone is the murine p110delta , since it shares 94% amino acid sequence identity with human p110delta (compared with 56% identity between human p110delta and beta ; an alignment of human and mouse p110delta is shown in Fig. 1) and has a similar pattern of expression in vivo (see below).

The sequences of human and murine p110delta include open reading frames predicted to encode for proteins of 1044 and 1043 amino acids, respectively, with an expected molecular mass of 119,505 Da for the human clone (~120 kDa). The sequences around the predicted initiating methionines are in good agreement with that required for optimal translational initiation (24). The presence of stop codons in the 5'-untranslated sequence is consistent with isolation of the complete coding region of p110delta (data not shown). Consistent with the predicted size of the encoded translation product, Western blotting of immunoprecipitates from COS cells transiently transfected with an epitope-tagged form of human p110delta detected a protein of ~110 kDa (see below).

Comparison of the sequence of the carboxyl-terminal catalytic domain of p110delta with those of other PI 3-kinases reveals that it is most closely related to p110beta . p110delta is 72% identical to p110beta in this region and is less closely related to p110alpha (49%) or p110gamma (45%), whereas cpk/p170 and the yeast Vps34 protein show the lowest homology (31 and 32%, respectively). This is confirmed by dendrogram analysis; p110beta and p110delta form a distinct sub-branch of the PI 3-kinase family (Fig. 2). The distantly related ataxia telangiectasia gene product and the catalytic subunit of DNA-dependent protein kinase have been included for comparison (Fig. 2). These proteins are structurally related to PI 3-kinases and have protein kinase activity (25) but have not yet been shown to possess lipid kinase activity (26, 27).


Fig. 2. Dendrogram analysis of the PI 3-kinase family. The analysis was performed based on the predicted catalytic domains corresponding to the C-terminal 320 amino acids. DNA PKCSCS, catalytic subunit of DNA-dependent protein kinase; ATM, ataxia telangiectasia gene product.
[View Larger Version of this Image (11K GIF file)]

p110alpha and p110beta form heterodimers with a common p85 subunit (3-5, 8). We examined the association of recombinant p110delta with p85. When either epitope-tagged human (Fig. 3, panel B) or mouse (Fig. 4, lane 4) p110delta was expressed in COS or 293T cells and recovered by immunoprecipiation, a 85-kDa protein was detected with p85-specific antiserum in the immunoprecipitates. Thus, both human and murine p110delta associate with endogenous p85 after transfection into COS or 293T cells, respectively. Association of human p110delta with p85 could also be detected following expression of epitope-tagged p110delta in the lymphoid cell line Jurkat (data not shown). The association of p110alpha , p110beta , and p110delta with p85 is consistent with the presence of a conserved p85 interaction domain at the amino terminus of these isoforms. This region is lacking in p110gamma , which is targeted to the plasma membrane via its interaction with the beta /gamma subunits of heterotrimeric G proteins. This interaction is dependent on a p110gamma -specific adaptor protein, p101 (28).


Fig. 3. Human p110delta associates with p85alpha . FLAG-tagged p110delta was expressed in COS cells and immunoprecipitated with the anti-FLAG M2 antibody. Immunoprecipitates were analyzed by 8% SDS-polyacrylamide gel electrophoresis followed by immunoblotting using either anti-FLAG M2 or anti-p85alpha . Panel A shows coimmunoprecipitation of p110delta with p85alpha . The control lane is a lysate from the Jurkat cell line that constitutively expresses p85alpha . Panel B shows that anti-FLAG M2 recognizes an ~110-kDa protein in immunoprecipitates from p110delta -transfected cells.
[View Larger Version of this Image (41K GIF file)]


Fig. 4. Murine p110delta associates with p85. Glutathione S-transferase-tagged murine p110delta was expressed in 293T cells, and the glutathione S-transferase fusion protein was recovered by incubation with glutathione-Sepharose resin. Proteins bound to the resin were analyzed by 12.5% SDS-polyacrylamide gel electrophoresis followed by immunoblotting using an anti-glutathione S-transferase antibody or anti-p85. Lanes 1 and 2 show expression of the fusion protein. Lanes 3 and 4 show that endogenous p85alpha associates with the glutathione S-transferase-tagged murine p110delta . An arrow marks the position of the p85 protein.
[View Larger Version of this Image (81K GIF file)]

It has been demonstrated that PI 3-kinase is an important intermediate in the Ras pathway (29, 30). A specific region at the amino terminus of the p110alpha subunit, residues 133-314, termed the Ras regulatory domain (underlined in Fig. 1), is responsible for this interaction (30). Comparison of the sequence of both human and murine p110delta with other p110 subunits indicates that this region is also conserved in p110delta , including a lysine residue (residues 227 of p110alpha and 223 of p110delta ), which has been shown to be essential for physical association with Ras (30). Moreover, a relatively short amino acid domain of p110delta (amino acids 141-291, the amino acids of p110delta encoded by the Rip36 clone) is sufficient to interact with Ha-RasV12 in vivo in the yeast two-hybrid system and further delineates the Ras regulatory domain to a 151-amino acid region. The interaction of Ha-RasV12 with this domain of p110delta requires active Ras and an intact Ras effector domain (data not shown). Thus, p110delta is likely to mediate some of the effects of Ras, although the p110delta Ras-interacting region is less conserved than the putative p85 binding site or catalytic domain.

Whereas the activation of PI 3-kinase in a wide range of biological systems has been extensively studied, less is known concerning the cell type-specific expression of particular p110 isoforms. Northern blot analysis of the expression of p110delta in human and murine tissues reveals a single transcript of ~5.4 kilobases (consistent with the size of the composite cDNAs). In humans, the highest levels of expression are seen in PBMC, spleen, and thymus (Fig. 5). After prolonged exposure of the autoradiograph, low levels of p110delta expression could be detected in testes, uterus, colon, and small intestine but not in other tissues examined including prostate, heart, brain, and liver (data not shown.) p110delta is also abundantly expressed in adult mouse spleen as well as in testis (Fig. 5B). The elevated expression of p110delta mRNA in mouse but not human testes is noteworthy and may reflect a true difference in expression between species. Alternatively, a number of genes is expressed specifically at elevated levels in postmeiotic haploid cells in the testis. Abnormally sized transcripts that may be more stable than the transcripts found in diploid cells are commonly found. However, it is not clear whether these transcripts are translated (Ref. 31 and references cited therein). In the case of p110delta , the RNA expressed in the testis may or may not be translated. If p110delta protein is expressed, then it is possible that the protein has a specific role in development of the male germ line. The restricted expression of p110delta is in contrast to p110alpha and p110beta , which appear to be widely expressed (3, 8).


Fig. 5. Tissue distribution of p110delta . RNA from various human or murine tissues was examined for the expression of p110delta by Northern blotting. A, human tissues hybridized with the human cDNA probe. B, murine tissues hybridized with the murine cDNA probe. H, heart; B, brain; S, spleen;, Lu, lung; M, muscle; K, kidney; T, testes; L, liver.
[View Larger Version of this Image (50K GIF file)]

To test whether p110delta has PI 3-kinase activity, immunoprecipitates from COS cells transfected with epitope-tagged p110delta were incubated with [32P]ATP and phosphatidylinositol, and the radiolabeled phospholipids were resolved by chromatography. A product was detected that migrates slightly slower than the PI 4-phosphate (PIP) standard, consistent with the generation of PI 3-phosphate (32) (Fig. 6). This enzyme activity was sensitive to the PI 3-kinase inhibitor wortmannin (data not shown). Similar results were obtained when purified phosphatidylinositol was used as a substrate (data not shown). Whereas these results demonstrate that the cDNA clone that we isolated encodes a functional PI 3-kinase, it cannot be assumed that the in vitro substrate specificity of a particular isoform reflects its activity on membrane lipids in intact cells (11).


Fig. 6. p110delta has PI 3-kinase activity. Anti-p110delta immunoprecipitates were assayed for PI 3-kinase activity using phosphatidylinositol as a substrate. Reactions were performed as described under "Materials and Methods." The reaction products were resolved by thin layer chromatography followed by autoradiography. PI 3-kinase activity is detected in immunoprecipitates from p110delta -transfected (but not vector control) cells. The position of the PI 4-phosphate (PIP) standard is shown. ORI, origin.
[View Larger Version of this Image (50K GIF file)]

The interaction of multiple p110 catalytic subunit isoforms, p110alpha , p110beta , and p110delta , with a common adaptor protein, p85, suggests that the nature of the phosphorylated lipids generated in response to a particular agonist may be regulated at least in part by the cell/tissue-specific expression of the different isoforms of the catalytic subunit. In cells such as leukocytes, where it is likely that multiple p110 isoforms are expressed, it will be of interest to determine the relative contribution made by these multiple isoforms to processes such as cell activation and migration.


FOOTNOTES

*   This work was supported by ICOS Corporation and by Public Health Service Grant CA54786 (to J. C.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U86587 and U86453.


par    Current address: Millenium Pharmaceuticals, Cambridge, MA 02139.
**   To whom correspondence should be addressed: ICOS Corp., 22021 20th Ave. S.E., Bothell WA 98021. Tel.: 206-485-1900, Fax: 206-485-1961.
1   The abbreviations used are: PI, phosphatidylinositol; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; PBMC, peripheral blood mononuclear cells; RT, reverse transcription; PIPES, 1,4-piperazinediethanesulfonic acid; Rip, Ras-interacting protein.

ACKNOWLEDGEMENTS

We thank David Turner, Bart Vanhaesebroeck, and Pablo Rodriguez-Viciana for discussions and Johnny Stine for help in preparing the figures.


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