©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Sequence of Phosphatidylinositol-4-phosphate 5-Kinase Defines a Novel Family of Lipid Kinases (*)

(Received for publication, December 6, 1994)

Igor V. Boronenkov Richard A. Anderson (§)

From the Departments of Pharmacology and Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) occupies an essential position in the phosphoinositide signal transduction cascades as the precursor to second messengers and is thought to regulate many cellular proteins directly. The final step in the synthesis of PtdIns(4,5)P(2) is the phosphorylation of PtdIns(4)P by PtdIns(4)P 5-kinase (PIP5K). Using peptide sequences from a purified PIP5K, a cDNA for a human placental PIP5K was isolated and sequenced. Expression of this cDNA in Escherichia coli produced an active PIP5K. Surprisingly, the sequence of this PIP5K has no homology to known PtdIns kinases or protein kinases. However, the PIP5K is homologous to the Saccharomyces cerevisiae proteins Fab1p and Mss4p.


INTRODUCTION

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (^1)directly modulates the in vitro activity of a number of enzymes, including protein kinase C, casein kinase I, and phospholipase D (1, 2, 3) . The activities of cytoskeletal proteins, profilin, gelsolin, protein 4.1, alpha-actinin, and others are also regulated by PtdIns(4,5)P(2)(4, 5, 6) . Recently, pleckstrin homology domains were found to bind PtdIns(4,5)P(2), and several proteins that contain these domains are involved in signal transduction(7) . PtdIns(4,5)P(2) is also a bifurcation point in the PtdIns signal transduction cascade(8, 9, 10, 11, 12) . PtdIns(4,5)P(2) can be hydrolyzed by receptor-activated phospholipase C enzymes(9) , generating inositol 1,4,5-trisphosphate, which stimulates intracellular Ca release(8) , and diacylglycerol, which activates some protein kinase C isoforms(10) . Alternatively, PtdIns(4,5)P(2) can be a substrate for receptor-stimulated PtdIns 3-kinases, which synthesize PtdIns(3,4,5)P(3), a second messenger of unknown function(11, 12) . The candidate functions of PtdIns(4,5)P(2) are thought to include regulation of secretion, cell proliferation, differentiation, and motility(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) . Given the roles of PtdIns(4,5)P(2), its synthesis is likely to be stringently regulated.

The first step in PtdIns(4,5)P(2) synthesis is the phosphorylation of PtdIns on the fourth hydroxyl of the myo-inositol ring by PtdIns 4-kinases(11) . Multiple PtdIns 4-kinases have been identified biochemically, and two PtdIns 4-kinase genes, PIK1 and STT4 have been characterized from Saccharomyces cerevisiae(11, 12, 13, 14, 15) . Pik1p is an essential nuclear associated enzyme(13, 14) . STT4 was identified as a staurosporine-sensitive mutant in the protein kinase C pathway(15) . Although STT4 is not essential, mutants grow slowly and have cell cycle defects similar to protein kinase C mutations(15) . The distinct phenotypes of these PtdIns 4-kinases suggest that they have different cellular roles.

PtdIns(4,5)P(2) synthesis is catalyzed by the phosphorylation of PtdIns(4)P on the fifth hydroxyl of the myo-inositol ring (11) . Two isoforms of PtdIns(4)P 5-kinase (PIP5K) have been characterized and are denoted type I and II PIP5Ks (PIP5KI and PIP5KII) (16, 17) . PIP5KI and PIP5KII are distinguished by their lack of immunocross-reactivity, by phosphatidic acid stimulation of PIP5KI, and by the low activity of purified PIP5KII toward native membranes(16, 17) . Both of these PIP5Ks use PtdIns(4)P as a substrate, and their product is PtdIns(4,5)P(2)(16, 17) .

Immunological evidence suggests that multiple PIP5KI and PIP5KII isoforms exist in different cell types and may have different functions (16, 17, 18, 19) . For example, the G-protein Rho stimulated a PIP5K, and this appeared to be required for integrin-dependent, platelet-derived growth factor-stimulated phosphoinositide turnover(20) . This suggests that regulation of PIP5K activity is more crucial for phosphoinositide signaling than was previously thought.

The phosphoinositides have been implicated directly in Ca-regulated secretion(3, 21) . (^2)One of the cytosolic factors required for ATP-dependent priming of Ca-regulated secretion corresponds to a PIP5KI.^2 Purified erythroid 68-kDa PIP5KI stimulated priming; however, a brain 90-kDa PIP5KIb (17) appeared to have greater priming activity.^2 Surprisingly, PIP5KII had no detectable activity in ATP-dependent priming of regulated secretion.^2

To assign regulatory mechanisms and cellular roles to individual PIP5Ks, isolation of the cDNAs and determination of the sequences of the PIP5Ks are critical. Here we report the isolation and characterization of the first cDNA encoding a PIP5K.


MATERIALS AND METHODS

Isolation of the PIP5KII cDNA

Human erythroid 53-kDa PIP5KII was purified (16) and digested overnight at 37 °C with Achromobacter lysine-specific protease (Wako Bioproducts). The peptides were purified by reverse-phase HPLC on a C(4) column and sequenced(23) . These sequences were searched against the GenBank Version 84.0 data base using the BLAST algorithm (24) . Two peptides aligned within the same reading frame of a putatively transcribed sequence from a human bone marrow gt11 cDNA library (GenBank accession number Z20468). This 548-bp cDNA was subcloned into pBluescript SK(-), sequenced (Sequenase Version 2.0 DNA sequencing kit, U. S. Biochemical Corp.), and used to screen a human placental gt11 cDNA library (CLONTECH). About 6.5 times 10^5 plaques were transferred to nitrocellulose filters and hybridized at 42 °C in 6 times saline/sodium phosphate/EDTA buffer, 5 times Denhardt's solution, 0.5% SDS, 50% formamide, and 100 mg/ml carrier DNA(25) ; this yielded two clones. The largest (1463 bp) was subcloned into pBluescript SK(-) and sequenced; this cDNA had an open reading frame of 405 amino acids, which codes PIP5KII. The cDNA sequence was determined on both strands by a combination of subclones and custom-made primers.

Expression in Escherichia coli

The full coding region of PIP5KII was subcloned into the NcoI site of the pRSET B vector (Invitrogen). Expression was induced by the bacteriophage CE6 (Novagen). Recombinant His-PIP5KII was purified from cleared E. coli strain JM109 lysates by chromatography on a Ni-charged chelate resin column (Novagen) according to the manufacturer's instructions. After chromatography, the protein samples were dialyzed against 50 mM Tris-HCl (pH 8.0) and 0.1 mM phenylmethylsulfonyl fluoride. PtdIns(4)P kinase was assayed for activity and Western blotted as described before(16, 17) .

Northern Blotting

A human multiple tissue Northern blot (CLONTECH) was hybridized according to the manufacturer's instructions with a random-primed, alpha-P-labeled 497-bp EcoRI fragment derived from the 3`-part of the coding region of the PIP5KII cDNA(25) . A 569-bp EcoRI fragment from the 5`-part of the coding region was also used to probe the same filter, as was a BalI-EcoRI probe from the 3`-region of the cDNA. Each lane contained 2 µg of poly(A) RNA.


RESULTS AND DISCUSSION

Isolation and Sequencing of a PIP5KII cDNA

Peptide sequences were obtained from purified human erythroid 53-kDa PIP5KII(16) . A search of the sequence data bases revealed that two peptide sequences aligned within the same reading frame of a putatively transcribed sequence from a human bone marrow cDNA library. This cDNA was obtained, sequenced, and used to screen a human placental gt11 cDNA library. The largest cDNA of 1463 bp was sequenced, yielding a 405-amino acid complete reading frame for PIP5KII (Fig. 1). All nine peptides sequenced (120 residues) aligned with the deduced protein's sequence with >95% identity overall, demonstrating that the cDNA encodes a PIP5KII or a highly related protein. From the sequence, the calculated molecular mass of PIP5KII is 46,600 Da. The calculated size is smaller than the 53 kDa estimated by SDS-PAGE(16) . This discrepancy appears to be due to anomalous mobility on SDS-PAGE since recombinant PIP5KII has a mobility on SDS-PAGE consistent with erythroid PIP5KII.


Figure 1: Nucleotide and predicted amino acid sequences for the coding region of the human 53-kDa PIP5KII cDNA. The first ATG start codon in the open reading frame is preceded by an in-frame termination codon and is within a Kozak consensus translation initiation sequence(26) . The existence of at least 60 additional nucleotides 5` to the cDNA sequence displayed is suggested by a 5`-rapid amplification of cDNA ends procedure on human placental 5`-rapid amplification of cDNA ends-ready cDNA (CLONTECH). The alignments of peptide sequences from 53-kDa PIP5KII are underlined. The putative SH3 domain-binding sites are boxed. The termination codon is denoted by an asterisk.



The cDNA Encodes a PIP5K with Properties Indistinguishable from PIP5KII

To confirm that the placental cDNA encodes a PIP5K, the cDNA was expressed in E. coli as a polyhistidine fusion protein. A protein of 57 kDa as determined by SDS-PAGE, which was immunoreactive with PIP5KII antibodies, was detected in lysates of induced E. coli cells expressing the placental PIP5KII cDNA, but not vector alone (Fig. 2A). The larger size of the recombinant protein on SDS-PAGE was due to the vector-derived polyhistidine tag and linker sequence. The recombinant PIP5KII fusion protein was purified to homogeneity by Ni-charged chelate chromatography (Fig. 2A). Both purified recombinant PIP5KII and E. coli lysates from cells expressing PIP5KII exhibit PtdIns(4)P kinase activity (Fig. 2B). Lysates from induced E. coli cells bearing vector alone lack kinase activity. Furthermore, the biochemical properties of the recombinant enzyme are indistinguishable from those of purified PIP5KII(16) . The recombinant enzyme can use GTP and ATP as phosphate donors, has a K(m) for PtdIns(4)P of 80 µM, and is inhibited by increasing the ionic strength (data not shown). This is consistent with the cloned enzyme belonging to the PIP5KII subclass(16) .


Figure 2: Purification and kinase activity of PIP5KII expressed in E. coli. A: left panel, bacterially expressed PIP5KII is recognized by polyclonal antibodies raised against human erythroid 53-kDa PIP5KII(16) . Shown is a Western blot with PIP5KII antibodies of purified human erythrocyte PIP5KII; E. coli lysates (14 µg) with vector alone(-) or vector containing the cDNA (+); and affinity-purified, polyhistidine-tagged recombinant PIP5KII (His-PIP5KII). Right panel, Coomassie blue-stained SDS-polyacrylamide gel of a total lysate of E. coli expressing His-PIP5KII and purified His-PIP5KII. B, PtdIns(4)P kinase activity of native erythrocyte PIP5KII and recombinant His-PIP5KII. Shown is the PtdIns(4,5)P(2) synthesized by purified erythroid PIP5KII (6 ng); clarified E. coli lysates (350 µg) from induced cells harboring the vector alone(-) or expressing PIP5KII (+); and affinity-purified, recombinant His-PIP5KII (6 ng). PIP(2), PtdIns(4,5)P(2).



Expression Pattern of PIP5KII mRNA in Human Tissues

A ubiquitously expressed, 4.1-kilobase mRNA, with high levels in brain, was detected by Northern blot analysis of human poly(A) RNA from different tissues using a 3`-portion of the coding region (Fig. 3) or the 3`-untranslated region of the PIP5KII cDNA as a probe (data not shown). This wide expression pattern suggests the general importance of the placental PIP5K isoform in cellular processes. Using a 5`-part of the coding region as a probe, an additional 6.3-kilobase RNA was detected largely in brain, heart, and muscle, indicating that a highly related message is expressed in these tissues (data not shown). Indeed, a BLAST data base search (23) with placental PIP5KII sequence yielded a transcribed sequence from a human infant brain cDNA library(27) . Sequencing of this partial cDNA clone identified an open reading frame with 74% identity to the amino acid sequence of placental PIP5KII, suggesting that it encodes a closely related PIP5K isoform. This finding confirms the previously reported existence of multiple PIP5K isoforms(16, 17, 18, 19) .


Figure 3: Northern blot analysis of human tissues. A human multiple tissue Northern blot (CLONTECH) was hybridized (25) with an alpha-P-labeled 497-bp EcoRI fragment derived from the coding region of the PIP5KII cDNA.



The PIP5KII Sequence Is Distinct from Other Kinases, but Yeast Homologues Were Identified

Known PtdIns 4- and 3-kinases share sequence homology over distinct domains, and some of these domains are also found in protein kinases(12, 13, 14, 15, 28, 29, 30, 31) , suggesting that these enzymes may have a similar phosphotransferase mechanism. Surprisingly, the PIP5KII sequence has no homology to kinase motifs, implying that its catalytic mechanism is distinct.

Searching the sequence data bases with BLAST(24) , we found a significant similarity to two S. cerevisiae gene products, Fab1p (^3)and Mss4p(32) , at their C-terminal ends (Fig. 4A). Within this region, the identity between PIP5KII and these proteins is 27 and 34%, respectively. If conservative amino acid replacements are taken into account, the similarity among the three proteins is close to 60% in this region. This sequence identity is reminiscent of that among diverse protein kinases and between PtdIns 3- and 4-kinases(12, 13, 14, 15, 28, 29, 30, 31) . This is illustrated by the clustering of conserved residues into several domains (Fig. 4B). These conserved domains may represent parts of the catalytic core.


Figure 4: Comparison of the amino acid sequence of human placental PIP5KII with those of S. cerevisiae Fab1p and Mss4p. A, schematic representation of the proteins. Homologies among PIP5KII, Mss4p, and Fab1p are shown by shading. B, comparison of the amino acid sequences within the PtdIns(4)P kinase homology domain in the full-length human placental isoform of PIP5KII, Mss4p (residues 330-779), and Fab1p (residues 1900-2278). The alignment was performed using the PileUp program (University of Wisconsin Genetics Computer Group, Madison, WI). Conserved residues are in boldface.



The glycine-rich motif of the homologues at residues 149-155 (Fig. 4B) resembles the phosphate-binding loop of protein kinases, heterotrimeric G-proteins, RecA, elongation factor Tu, adenylate kinase, and H-ras(30) . It is also similar to the GTP-binding sequences in dynamin, Mx1, and Vps1p(31) . Since PIP5Ks can use ATP and GTP as phosphate donors(16) , this homology is consistent. Other conserved regions have no clear homology to kinases.

PIP5KII Contains a Putative src Homology 3 (SH3) Domain-binding Sequence

A noteworthy feature of PIP5KII is the two proline-rich sequences between residues 307 and 329 (Fig. 1). The spacing of these proline residues is identical to that of SH3 domain-binding consensus sequences(33, 34) . The proline-rich region has little similarity between PIP5KII and Mss4p and is missing in Fab1p (Fig. 4). If these putative SH3 domain-binding sequences are functional, it suggests mechanisms for PIP5KII regulation and the coupling of PtdIns(4,5)P(2) production to its utilization by PtdIns 3-kinases and phospholipases C. In vivo, both phospholipase C- and bovine PtdIns 3-kinase appear to selectively use PtdIns(4,5)P(2), while in vitro, PtdIns(4)P and PtdIns are also used as substrates(8, 9, 10, 11, 12) . Both of these enzymes contain SH3 domains and could interact with the putative SH3 domain-binding region in PIP5KII. Furthermore, the binding of SH3 domains to dynamin and PtdIns 3-kinase stimulates their activities(34, 35) . SH3 domains may bind and similarly modulate PIP5KII activity.

FAB1 and MSS4 May Encode PtdIns(4)P Kinases

The genetic data suggest that Mss4p is also a PtdIns(4)P kinase. MSS4 is an essential S. cerevisiae gene that was identified as a partial suppressor of a stt4 staurosporine-sensitive PtdIns 4-kinase mutation(32) . The STT4 PtdIns 4-kinase appears to be a component of the protein kinase C pathway in yeast. Furthermore, the part of the open reading frame of MSS4 with homology to placental PIP5KII was shown to be necessary for suppression of the stt4 mutant phenotype. Most important, MSS4 did not suppress the stt4 deletion mutant phenotype. This suggested that MSS4 works in a pathway downstream of STT4, which is consistent with Mss4p being a PtdIns(4)P kinase. The inability of Mss4p overexpression to completely suppress a G(2)/M boundary blockage phenotype of stt4 has been proposed to reflect a branch point in the pathway between STT4 and MSS4(32) . Thus, there may be a distinct pathway affecting G(2)/M cell cycle progression, which utilizes a PtdIns(4)P pool different from that used by Mss4p.

The S. cerevisiaeFAB1 gene was isolated based on the fab1 mutant phenotype, in which cells have profoundly enlarged vacuoles that are not acidified.^3 These mutants also have defective chromosomal segregation and abnormal spindle morphology. A chromosomal segregation defect is also observed upon mutation of the S. cerevisiaePLC1 gene, which encodes a phosphoinositide-specific phospholipase C(36, 37, 38) . However, there is no reported phenotypic changes of the vacuoles in PLC1 mutants. The presence of the PLC1 gene in S. cerevisiae suggests that MSS4 and FAB1 may be required for second messenger production (36, 37, 38) . MSS4 and FAB1 could also regulate other events, such as cytoskeletal assembly, by an interaction of PtdIns(4,5)P(2) with the Pfy1p profilin homologue (22) or other proteins or enzymes that have not yet been described.

The mammalian PIP5KI and PIP5KII isoforms are structurally and apparently functionally distinct(16, 17, 18, 19, 20) .^2 The different phenotypes associated with mutations of the yeast PIP5K homologues and the fact that MSS4 is essential, whereas FAB1 is not, suggest that these proteins in S. cerevisiae have diverse cellular roles, similar to the PtdIns 3- and 4-kinases. If this hypothesis is correct, then the PIP5K family of enzymes will likely be as large and diverse as the PtdIns 3- and 4-kinase families. The most striking feature of this PIP5K is its lack of sequence homology to known protein or PtdIns kinases.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM51968 and GM38906 (to R. A. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) U14957[GenBank].

§
To whom correspondence should be addressed: Depts. of Pharmacology and Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-3753; Fax: 608-262-1257.

(^1)
The abbreviations used are: PtdIns(4,5)P(2), phosphatidylinositol 4,5-bisphosphate; PIP5K, PtdIns(4)P 5-kinase; HPLC, high pressure liquid chromatography; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; SH3, src homology 3.

(^2)
J. C. Hay, P. F. Fisette, G. H. Jenkins, R. A. Anderson, and T. M. J. Martin, submitted for publication.

(^3)
A. Yamamoto and D. Koshland, personal communication.


ACKNOWLEDGEMENTS

We thank M. Sussman for HPLC separation of peptides; the University of Wisconsin Biotechnology Center for peptide sequencing; A. Yamamoto and D. Koshland for sharing unpublished data on FAB1; the United Kingdom Human Genome Mapping Project for providing the human bone marrow cDNA clone; T. F. J. Martin, W. Heideman, and J. Loijens for comments on the manuscript; and W. Wang for technical assistance.

Note Added in Proof-The product of PIP kinase reaction with recombinant enzyme was unambiguously shown to be PtdIns(4,5)Pz by alkaline hydrolysis, followed by HPLC analysis.


REFERENCES

  1. Lee, M., and Bell, R. M. (1991) Biochemistry 30, 1041-1049 [Medline] [Order article via Infotrieve]
  2. Brockman, J. L., and Anderson, R. A. (1991) J. Biol. Chem. 266, 2508-2512 [Abstract/Free Full Text]
  3. Liscovitch, M., Chalifa, V., Pertile, P., Chen, C.-S., and Cantley, L. C. (1994) J. Biol. Chem. 269, 21403-21406 [Abstract/Free Full Text]
  4. Anderson, R. A., and Marchesi, V. T. (1985) Nature 318, 295-298 [Medline] [Order article via Infotrieve]
  5. Fukami, K., Endo, T., Imamura, M., and Takenawa, T. (1994) J. Biol. Chem. 269, 1518-1522 [Abstract/Free Full Text]
  6. Janmey, P. A. (1994) Annu. Rev. Physiol. 56, 169-191 [CrossRef][Medline] [Order article via Infotrieve]
  7. Harlan, J. E., Hajduk, P. J., Yoon, H. S., and Fesik, S. W. (1994) Nature 371, 168-170 [CrossRef][Medline] [Order article via Infotrieve]
  8. Berridge, M. J. (1987) Annu. Rev. Biochem. 56, 159-193 [CrossRef][Medline] [Order article via Infotrieve]
  9. Rhee, S. G., and Choi, K. D. (1992) J. Biol. Chem. 267, 12393-12396 [Free Full Text]
  10. Nishizuka, Y. (1988) Nature 334, 661-662 [CrossRef][Medline] [Order article via Infotrieve]
  11. Carpenter, C. L., and Cantley, L. C. (1990) Biochemistry 29, 11147-11156 [Medline] [Order article via Infotrieve]
  12. Stephens, L. R., Jackson, T. R., and Hawkins, P. T. (1993) Biochim. Biophys. Acta 1179, 27-75 [Medline] [Order article via Infotrieve]
  13. Flanagan, C. A., Schnieders, E. A., Emerick, A. W., Kunisawa, R., Admon, A., and Thorner, J. (1993) Science 262, 1444-1448 [Medline] [Order article via Infotrieve]
  14. Garcia-Bustos, J. F., Marine, F., Stevenson, I., Frei, C., and Hall, M. N. (1994) EMBO J. 13, 2352-2361 [Abstract]
  15. Yoshida, S., Ohya, Y., Goebl, M., Nakano, A., and Anraku, Y. (1994) J. Biol. Chem. 269, 1166-1171 [Abstract/Free Full Text]
  16. Bazenet, C. E., Ruiz-Ruano, A., Brockman, J. L., and Anderson, R. A. (1990) J. Biol. Chem. 265, 18012-18022 [Abstract/Free Full Text]
  17. Jenkins, G. H., Fisette, P. L., and Anderson, R. A. (1993) J. Biol. Chem. 269, 11547-11554 [Abstract/Free Full Text]
  18. Payrastre, B., Nievers, M., Boonstra, J., Breton, M., Verkleij, A. J., and Van Bergen en Henegouwen, P. M. P. (1992) J. Biol. Chem. 267, 5078-5084 [Abstract/Free Full Text]
  19. Brooksbank, C. E. L., Hutchings, A., Butcher, G. W., Irvine, R. F., and Divecha, N. (1993) Biochem. J. 291, 77-82 [Medline] [Order article via Infotrieve]
  20. Chong, L. D., Kaplan, A. T., Bokoch, G. M., and Schwartz, M. A. (1994) Cell 79, 507-513 [Medline] [Order article via Infotrieve]
  21. Hay, J. C., and Martin, T. F. J. (1994) Nature 366, 572-575
  22. Magdolen, V., Oechsner, U., Müller, G., and Brandlow, W. (1988) Mol. Cell. Biol. 8, 5108-5115 [Medline] [Order article via Infotrieve]
  23. Brockman, J. L., Gross, S. D., Sussman, M. R., and Anderson, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9454-9458 [Abstract]
  24. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  25. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Kozak, M. (1991) J. Cell Biol. 115, 887-903 [Abstract]
  27. Adams, M. D., Kerlavage, A. R., Fields, C., and Venter, J. C. (1993) Nature Genetics 4, 256-267 [Medline] [Order article via Infotrieve]
  28. Herman, P. K., Stack, J. H., and Emr, S. D. (1992) Trends Cell Biol. 2, 363-368 [Medline] [Order article via Infotrieve]
  29. Hiles, I. D., Otsu, M., Volinia, S., Fry, M. J., Gout, I., Dhand, R., Panayotou, G., Ruiz-Larrea, F., Thompson, A., Totty, N. F., Hsuan, J. J., Courtneidge, S. A., Parker, P. J., and Waterfield, M. D. (1992) Cell 70, 419-429 [Medline] [Order article via Infotrieve]
  30. Bossemeyer, D. (1994) Trends Biochem. Sci. 19, 201-205 [CrossRef][Medline] [Order article via Infotrieve]
  31. Staeheli, P., Pitossi, F., and Pavlovic, J. (1993) Trends Cell Biol. 3, 268-272 [CrossRef]
  32. Yoshida, S., Ohya, Y., Nakano, A., and Anraku, Y. (1994) Mol. & Gen. Genet. 242, 631-640
  33. Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A. W., and Schrieber, S. L. (1994) Cell 76, 933-945 [Medline] [Order article via Infotrieve]
  34. Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J., Booker, G. W., Campbell, I. D., and Waterfield, M. D. (1993) Cell 75, 25-36 [Medline] [Order article via Infotrieve]
  35. Pleiman, C. M., Hertz, W. M., and Cambier, J. C. (1994) Science 263, 1609-1612 [Medline] [Order article via Infotrieve]
  36. Yoko-o, T., Matsui, Y., Yagisawa, H., Nojima, H., Uno, I., and Toh-e, A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1804-1808 [Abstract]
  37. Flick, J. S., and Thorner, J. (1993) Mol. Cell. Biol. 13, 5861-5876 [Abstract]
  38. Payne, W. E., and Fitzgerald-Hayes, M. (1993) Mol. Cell. Biol. 13, 4351-4364 [Abstract]

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