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Interaction of Activator of G-protein Signaling 3 (AGS3) with LKB1, a Serine/Threonine Kinase Involved in Cell Polarity and Cell Cycle Progression

Interaction of Activator of G-protein Signaling 3 (AGS3) with LKB1, a Serine/Threonine Kinase... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 26, Issue of June 27, pp. 23217–23220, 2003 Accelerated Publication © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. where both proteins are involved in cell polarity. LKB1 Interaction of Activator of immunoprecipitates from COS7 cells transfected with G-protein Signaling 3 (AGS3) LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR with LKB1, a Serine/Threonine domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phos- Kinase Involved in Cell Polarity phate moiety within a consensus GPR motif reduced the and Cell Cycle Progression ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may PHOSPHORYLATION OF THE G-PROTEIN be a general mechanism regulating the interaction of REGULATORY (GPR) MOTIF AS A REGULATORY GPR-containing proteins with G-proteins. Such a mech- MECHANISM FOR THE INTERACTION OF GPR anism may be of particular note in regard to localized MOTIFS WITH G * signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regu- Received for publication, December 11, 2002, lated by heterotrimeric G-proteins that occur independ- and in revised form, April 24, 2003 ently of a typical G-protein-coupled receptor. Published, JBC Papers in Press, April 28, 2003, DOI 10.1074/jbc.C200686200 Joe B. Blumer‡§, Michael L. Bernard¶, AGS3 was identified in a functional screen for receptor- Yuri K. Peterson‡, Jun-ichi Nezu**, Peter Chung¶, independent activators of G-protein signaling (1, 2). Surpris- Dara J. Dunican‡‡, Juergen A. Knoblich‡‡, ingly the activation of G-protein signaling in the functional and Stephen M. Lanier‡§§ screen was independent of nucleotide exchange on the G sub- From the **Chugai Pharmaceutical Co., Ltd., Tsukuba unit suggesting unexpected mechanisms for regulating the ac- Research Laboratory, 153-2 Nagai Niihari, Ibaraki tivation state of heterotrimeric G-proteins. AGS3 interacts 300-4101, Japan, the ‡‡Research Institute of Molecular Pathology (IMP), Dr. Bohr Gasse 7, A-1030 Vienna, with G-proteins (G /G ) via its four G-protein regulatory (GPR) i o Austria, the ¶Department of Pharmacology, Medical or GoLoco motifs, each of which interacts with G (G  G ) and i o University of South Carolina, Charleston, South stabilizes the GDP-bound conformation of G (3). The GPR Carolina 29425, and the ‡Department of Pharmacology motif is also found in other proteins including LGN, RGS12, and Experimental Therapeutics, Louisiana State RGS14, Rap1GAPII, Pcp2, and G18.1b (1, 4). AGS3 also contains University Health Sciences Center, New Orleans, seven tetratricopeptide repeats (TPRs) in the first half of the Louisiana 70112 protein, and these domains may serve as a regulatory domain for Activator of G-protein signaling 3 (AGS3) has a modu- the GPR-G-protein interaction, or they may target the protein to lar domain structure consisting of seven tetratricopep- different microdomains within the cell (5). A similar motif struc- tide repeats (TPRs) and four G-protein regulatory (GPR) ture is found in the AGS3-related protein LGN in mammals (6, 7) motifs. Each GPR motif binds to the  subunit of G /G i o as well as in the AGS3/LGN ortholog Pins in Drosophila mela- (G  > G ) stabilizing the GDP-bound conformation of i o nogaster, which is a key determinant of cell polarity (8 –12). A G and apparently competing with G for G bind- GDP role for GPR-containing proteins and G-proteins in cell polarity is ing. As an initial approach to identify regulatory mech- also suggested by studies in Caenorhabditis elegans (13, 14). anisms for AGS3-G-protein interactions, a yeast two-hy- AGS3 and other accessory proteins (proteins distinct from brid screen was initiated using the TPR and linker receptors, G-proteins, and effectors) may influence receptor- region of AGS3 as bait. This screen identified the serine/ mediated signaling events and/or mediate signal input to G- threonine kinase LKB1, which is involved in the regula- tion of cell cycle progression and polarity. Protein inter- proteins independently of a G-protein coupled receptor. Such action assays in mammalian systems using transfected proteins may also serve as alternative binding partners for cells or brain lysate indicated the regulated formation of G-protein subunits independently of heterotrimer formation (1, a protein complex consisting of LKB1, AGS3, and G- 2, 15), and the existence of these accessory proteins suggests proteins. The interaction between AGS3 and LKB1 was unexpected functional roles for G-proteins within the cell. As also observed with orthologous proteins in Drosophila an initial approach to define the cellular control mechanisms for AGS3-G-protein interactions, we sought to identify binding * This work was supported in part by National Institutes of Health partners for the TPR domains of AGS3. Grants MH90531 (to S. M. L.), NS24821 (to S. M. L.), and F32MH65092 We isolated several candidate AGS3-TPR-interacting pro- (to J. B. B.). The costs of publication of this article were defrayed in part teins in a yeast two-hybrid screen, one of which corresponded to by the payment of page charges. This article must therefore be hereby the carboxyl-terminal 107 amino acids of LKB1, also known as marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. serine/threonine kinase 11 (STK11) (16). Loss of LKB1 is im- § Recipient of an individual National Research Service Award sup- plicated in Peutz-Jeghers syndrome, a rare inherited intestinal ported by National Institutes of Health Grant F32MH65092. Recipient of a Medical Scientist Training Program Fellowship sup- ported by National Institutes of Health Grant T32-GM08716. The abbreviations used are: AGS3, activator of G-protein signaling §§ Recipient of a Research Scholar Award from Yamanouchi Phar- 3; TPR, tetratricopeptide repeat; GPR, G-protein regulatory; Pins, Part- maceutical Company, Inc. and the David R. Bethune/Lederle Labora- ner of Inscuteable; Ade, adenine; GST, glutathione S-transferase; tories Professor of Pharmacology. To whom correspondence should be MACF, microtubule/actin cross-linking factor; STK11, serine/threonine addressed: Dept. of Pharmacology, Louisiana State University Health kinase 11; CT, carboxyl terminus; GTPS, guanosine 5-3-O-(thio)tri- Sciences Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504- phosphate; Dm, Drosophila melanogaster; RGS, regulator of G-protein 568-4740; E-mail: [email protected]. signaling; GAP, GTPase-activating protein. This paper is available on line at http://www.jbc.org 23217 This is an Open Access article under the CC BY license. 23218 LKB1 and Phosphorylation of AGS3-GPR Domain polyposis syndrome (16 –18), and LKB1 is actually the mam- malian counterpart of the C. elegans gene par-4, which is a member of a group of polarity-determining genes during em- bryogenesis in both C. elegans and Drosophila (19 –21). Signif- icantly LKB1 phosphorylates AGS3 in its GPR domain, and this was completely blocked by a consensus GPR motif peptide. Place- ment of a phosphate moiety within a consensus GPR motif mark- edly reduced the ability of the peptide to interact with G-proteins suggesting that phosphorylation of GPR motifs may be a general mechanism regulating the interaction of GPR-containing pro- teins with G-proteins. Such a mechanism may be of particular note in regard to the localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur in- dependently of a typical G-protein coupled receptor. EXPERIMENTAL PROCEDURES FIG.1. Interaction of LKB1 with AGS3. A, rat brain (2 mg) lysate was preincubated with 30 M GDP or 30 M GTPS, 25 mM MgCl at Materials—Yeast strains pretransformed with prey libraries, c-Myc 24 °C for 30 min. Lysates were then incubated with 500 nM GST or monoclonal antiserum, and KC-8 chemically competent cells were ob- 330 436 GST-LKB1-CT (Asp –Gln )for1hat24 °C. Protein complexes were tained from Clontech (Palo Alto, CA). Bait vector pGBKT7 and yeast captured by glutathione-Sepharose beads and analyzed by immunoblot- strains Y187 and AH109 were kindly provided by Dr. Tim McQuinn ting following SDS-PAGE. Membrane transfers were first blotted with 32 32 (Medical University of South Carolina). [ - P]ATP and [ P]orthophos- AGS3 antiserum and then stripped and reprobed with G  antiserum. i 3 phate were obtained from PerkinElmer Life Sciences. Sodium or- The Input lane contains ⁄10 of the lysate volume used for each interac- thovanadate and RNase A were obtained from Sigma. Okadaic acid was tion assay. The data are representative of two experiments. B, LKB1 obtained from Calbiochem. pMAL-c2x and amylose-agarose beads were co-immunoprecipitates AGS3. COS7 cells transiently transfected with obtained from New England Biolabs (Boston, MA). Other materials empty vector (V), pcDNA3::AGS3, or pcDNA3::AGS3  pcDNA3::LKB1 were obtained as described elsewhere (3, 7). were lysed in Nonidet P-40 buffer prior to immunoprecipitation with 1 462 Yeast Two-hybrid Screening—AGS3-TPR (Met –Ile ) was gener- LKB1-specific antiserum and immunoblotting with AGS3-specific ated by PCR. Restriction enzyme-digested PCR products were sub- (PEP32) and LKB-specific (P6) antisera. C, Drosophila AGS3 ortholog cloned into pGBKT7 to generate the TPR bait construct. TPR and Pins interacts with Drosophila LKB1 in embryo lysates. Drosophila empty pGBKT7 vector were transformed into AH109 by the lithium embryo lysates were immunoprecipitated with DmLKB1 or Pins anti- acetate method. Expression of bait fusion proteins was confirmed by serum and association of these two proteins was evaluated by SDS- PAGE and immunoblotting for Pins (top panel) and DmLKB1 (bottom immunoblotting with anti-c-Myc. Basal activity of bait strains was panel). The input lane represents ⁄40 of the lysate volume used for each assayed by nutritional selection. AH109 yeast strains expressing TPR immunoprecipitation. IP, immunoprecipitation. as bait were mated with Y187 yeast strains expressing an 11-day-old mouse embryo cDNA library by following the manufacturer’s protocol. The mated yeast culture was plated onto 120 quadruple dropout rabbit anti-Pins (1:100). Mouse anti-Drosophila LKB1 was generated (Trp Leu His Ade ) plates that were then incubated at 30 °C for 7 against a maltose-binding protein-LKB1 fusion protein and recognizes days. -Galactosidase activity was screened using the colony-lift filter a single 75-kDa band in embryonic extracts. Rabbit anti-Pins was assay according to the manufacturer’s directions using diploid p53/ described before (11). SV40 large T antigen interaction (diploid strain PJ69-2A[pVA3-1] Kinase Assay—LKB1 immunoprecipitates were washed three times Y187[pTD1-1]) as a positive control as supplied by the manufacturer. with Nonidet P-40 lysis buffer and three times with kinase buffer A (50 Yeast plasmid DNA was isolated and used to transform competent mM Tris, pH 7.5, 0.1% -mercaptoethanol, 0.1 mM EGTA, 10 mM MnCl , KC-8 Escherichia coli cells. Transformants containing the prey vector 0.5 M okadaic acid) (17). The Gamma-Bind Sepharose was then resus- were selected by plating onto M9 Leu plates. Plasmids isolated from pended in kinase buffer A containing 10 M [- P]ATP (1000 cpm/pmol) KC-8 transformants were transformed into XL1-Blue E. coli cells for and 1 M purified GST fusion protein. Reactions were incubated at further processing and retransformation of yeast strains. 30 °C for 1 h. The Gamma-Bind Sepharose beads were pelleted, and the Immunoprecipitation and Cell Labeling—100-mm dishes of conflu- supernatant was removed and incubated with glutathione-Sepharose ent COS7 cells were transfected with either 10 g of empty vector (Amersham Biosciences) for 30 min at 24 °C to isolate the GST fusion (pcDNA3), 5 g of pcDNA3::AGS3  5 g of empty vector, or 5 gof proteins. The glutathione-Sepharose was pelleted and washed three pcDNA3::AGS3  5 g of pcDNA3::LKB1 (mouse). After 24 h, cells were times with kinase buffer A, resuspended in 5 protein sample buffer, lysed in Nonidet P-40 lysis buffer and incubated on ice for 1 h. The and placed in a boiling water bath for 3 min followed by SDS-PAGE and lysate was centrifuged at 100,000  g for 30 min at 4 °C and precleared autoradiography. with Gamma-Bind Sepharose (Amersham Biosciences). The precleared RESULTS AND DISCUSSION lysates (1 mg of protein) were incubated with 5 g of anti-LKB1 (Up- state Biotechnology, Inc., Lake Placid, NY) for 12–18hat4 °C. Gamma- Although AGS3 clearly interacts with G-proteins and the Bind Sepharose was added, and incubation continued for 30 min. The GPR motifs in AGS3 and other GPR-containing proteins stabi- resin was pelleted and used for kinase assays. Samples were washed lize the GDP-bound conformation of G, only a subpopulation three times with Nonidet P-40 lysis buffer, resuspended in 5 protein of AGS3 and G-proteins are associated with each other in brain sample buffer, and placed in a boiling water bath for 3 min followed by SDS-PAGE and immunoblotting with AGS3-specific (PEP32) (3) and lysates (3), and the two proteins exhibit minimal overlap in LKB-specific (P6) (22) antisera. PC12 cells were labeled with terms of their subcellular distribution (5). These data suggest [ P]orthophosphate (8500 –9120 Ci/mmol) according to Kang et al. (23) that the interaction between AGS3 and G-proteins is a regu- except that phosphate-free Dulbecco’s modified Eagle’s medium was used. lated event. As part of a broader strategy to address this issue For experiments with the Drosophila proteins, 3.5–7-h-old wild-type and define the role of AGS3-G-protein interactions in cellular Drosophila embryos were collected, dechorionated, and lysed by Dounce function, we used a protein interaction screen to identify bind- homogenization in ice-cold embryo lysis buffer (25 mM Tris-HCl, pH 8.0, 27.5 mM NaCl, 20 mM KCl, 25 mM sucrose, 10 mM EDTA, 10 mM EGTA, ing partners for the TPR and linker region of AGS3. A yeast 1mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10% glycerol, two-hybrid screen of a mouse 11-day-old embryonic cDNA li- 0.1% Nonidet P-40 supplemented with protease and phosphatase inhib- 1 462 brary using AGS3-TPR (Met –Ile ) as bait yielded several itors). The resultant lysate was centrifuged at 14,000 rpm in a 4 °C candidate AGS3 binding partners. The screen was run with bench-top centrifuge, and the supernatants were used for immunopre- high stringency by directly using quadruple dropout selection cipitation. Each immunoprecipitation (1:60 dilution of antisera) used (Trp Leu His Ade ) followed by a secondary selection for col- lysate corresponding to 150 l of packed embryos in a total volume of 300 l. Immunoblots were performed with mouse anti-LKB1 (1:200) or onies that exhibited strong -galactosidase activity within 30 LKB1 and Phosphorylation of AGS3-GPR Domain 23219 FIG.3. Influence of GPR phosphorylation on G-protein inter- action. A, protein interaction assays were performed as described under “Experimental Procedures” using 75 nM G  and 300 nM GST- i 1 AGS3 in the presence of 10 M GDP. Peptide concentration  10 M. Similar results were obtained in three separate experiments. The Input lane represents ⁄10 of the total volume in each interaction assay. B, GTP S (500 nM) binding to G  (100 nM) was measured after incuba- tion with increasing concentrations of control GPR consensus peptide and a phosphorylated Ser (PhosphoS16) GPR peptide as described under “Experimental Procedures.” Protein interaction assays and GTPS binding assays were performed as described previously (28). Data are expressed as the percentage of specific binding (0.5 pmol) FIG.2. LKB1 phosphorylates the GPR domain of AGS3. A, top observed in the absence of added peptide and are expressed as the mean  S.E. of two experiments with duplicate determinations. panel, COS7 cells were transfected with 10 g of pcDNA3::LKB1 or empty vector for 24 h prior to immunoprecipitation and immunoblotting with LKB1 antiserum. Bottom panel, purified GST fusion proteins (1 The cDNA clone encoding LKB1 contained the last 107 g/lane) were subjected to SDS-PAGE and Coomassie Blue staining. amino acids of the coding region of LKB1. The interaction of Right panel, LKB1 immunoprecipitates from LKB1-transfected COS7 1 462 1 462 AGS3-TPR (Met –Ile ) with LKB1 in the yeast two-hybrid cells were incubated with 1 M GST, GST-AGS3-TPR (Met –Ile ), or 338 462 463 650 32 GST-AGS3-GPR (Pro –Ser ) in the presence of [- P]ATP for1hat screen required amino acids Asp –Ile in the AGS3 coding 30 °C as described under “Experimental Procedures.” GST fusion pro- region that connects the TPR and GPR domains. Additional teins were purified with a glutathione resin prior to SDS-PAGE and regions of AGS3 and LKB1 may also interact with each other in autoradiography. The data are representative of three experiments. B, 3,4 the context of the full-length proteins. We then asked whether LKB1 kinase assays were performed as described under “Experimental 463 650 Procedures” with GST or GST-AGS3-GPR (Pro –Ser ) fusion pro- the interaction between the carboxyl terminus of LKB1 and teins in the presence or absence of a consensus GPR peptide (TMGEED- AGS3 was observed in a mammalian system using a GST fusion FFDLLAKSQSKRMDDQRVDLAG) or a scrambled control peptide protein of the LKB1 fragment isolated in the yeast two-hybrid (TMGDDQRLLAKSQSKRMEEDFFDVDLAG) at a final concentration 330 436 screen. LKB1-CT (Asp –Gln ) effectively interacted with en- of 100 M. GST fusion proteins were purified with glutathione-Sepha- dogenous, full-length AGS3 in rat brain lysates (Fig. 1). Interest- rose, washed, and subjected to SDS-PAGE and autoradiography. Data are representative of two experiments. C, PC12 cells, which express ingly this complex also contained G  subunits, which is likely endogenous AGS3, were labeled with [ P]orthophosphate 3 h prior to due to an interaction of G-proteins with the GPR motifs of AGS3 cell lysis (500 g of protein) and immunoprecipitated with AGS3 (3). The presence of G-proteins in this complex was nucleotide- antiserum followed by SDS-PAGE and autoradiography. Parallel unla- dependent in that it was not observed in the presence of the beled samples (500 g of protein) were used for immunoblotting with anti-AGS3 antiserum to confirm expression and immunoprecipitation. nonhydrolyzable GTP analog GTPS, which is consistent with Data are representative of two experiments. IP, immunoprecipitation; the demonstrated preference of GPR motifs for the GDP-bound IB, immunoblotting; autorad, autoradiography. conformation of G (1, 3, 28). The interaction of AGS3 itself with LKB1 was not influenced by guanine nucleotides. min. Sixteen cDNAs were isolated, five of which encoded DNA- The LKB1-AGS3 interaction was further addressed with the binding proteins or proteins involved in regulation of transcrip- full-length proteins in the intact cell. Co-transfection of cDNAs tion or translation. Of the remaining 11, one cDNA clone en- encoding full-length AGS3 and LKB1 in COS7 cells and sub- coded an extracellular protein, and five encoded previously sequent immunoprecipitation with LKB1 antiserum resulted unidentified proteins or proteins of unknown function. The in co-immunoprecipitation of AGS3 (Fig. 1B). This interaction remaining cDNA clones encoded portions of murine robo-1, an was specific for LKB1 as immunoprecipitation with LKB1 an- axonal guidance receptor during central nervous system devel- tiserum from cells transfected with AGS3 alone did not co- opment (24); microtubule/actin cross-linking factor (MACF, immunoprecipitate AGS3 (Fig. 1B). In contrast to the results also known as ACF7), a member of the plakin family implicated 330 436 obtained with the GST-LKB1-CT (Asp –Gln ) fusion pro- in epithelial and neuronal polarity (25); MARCKS (myristo- tein in which G  was brought down with the LKB1-CTAGS3 ylated alanine-rich C kinase substrate)-like protein, a regula- complex from brain lysates, G  was not found in the co-immu- tor of actin dynamics, migration, and neuronal development noprecipitation complex of the full-length proteins suggesting (26); and LKB1/STK11 (27). LKB1 and the Drosophila AGS3/ that LKB1 may process incoming signals to regulate the inter- LGN ortholog Pins are both involved in various aspects of cell action between AGS3 and G-proteins or target the protein to a polarity and development. As a tertiary screen to select for microdomain where G-proteins are inaccessible. proteins of potential interest, we asked whether GST fusion To provide further evidence for a functional interaction be- proteins of each of these cDNA clones interacted with full- tween LKB1 and AGS3, we asked whether the interaction was length AGS3 in brain lysates. Only GST-MACF and GST-LKB1 evolutionarily conserved in Drosophila. Drosophila LKB1 effectively pulled down AGS3 from rat brain lysates. We first (DmLKB1) co-immunoprecipitated with the AGS3 ortholog focused our effort on LKB1 as the LKB1 ortholog in C. elegans Pins, and conversely Pins co-immunoprecipitated with was previously identified as a PAR (partitioning defective) gene DmLKB1 in Drosophila embryo lysates, indicating an interac- (19) involved in asymmetric division of C. elegans embryos. AGS3/LGN orthologs or proteins containing GPR motifs are also J. B. Blumer, Y. K. Peterson, and S. M. Lanier, unpublished involved in similar events in Drosophila and C. elegans. observations. AGS3 was also isolated in a yeast two-hybrid screen using LKB1 as M. L. Bernard, J. B. Blumer, and S. M. Lanier, unpublished bait, and both proteins were reported to be involved in astrocyte polar- observations. ity (A. Hall, personal communication). 23220 LKB1 and Phosphorylation of AGS3-GPR Domain tion of the full-length Drosophila proteins (Fig. 1C). In both the within the GPR motif. GPR motifs are found in several proteins COS7 transfectants and the Drosophila embryos only a sub- involved in signal propagation including LGN, RGS12, RGS14, population of AGS3 (or Pins) was actually complexed with Rap1GAP, Pcp2, and G18.1b (1, 4). A recent report also sug- LKB1 following immunoprecipitation. This may reflect the af- gested that phosphorylation near the GPR motif of RGS14 may finity of the interaction, stoichiometric considerations, and/or influence GPR-G  interaction (32). Such a mechanism may regulation of the interaction by an as yet undefined signal(s). The allow the discrete regulation of G-protein signaling in specific AGS3 ortholog in Drosophila plays a critical role in cell polarity subcellular compartments that occurs independently of a G- that apparently also involves heterotrimeric G-proteins (8 –12). protein-coupled receptor. The localized regulation of such The LKB1 ortholog in Drosophila was also recently identified in events is a signature mechanism for the determination of cell a genetic screen for defects in oocyte and epithelial cell polarity polarity and asymmetric cell division observed in stem cells (21). The demonstration of an interaction between LKB1 and during tissue development. AGS3/LGN orthologs in Drosophila provides additional evidence Acknowledgments—We thank Dr. Thomas W. Gettys (Pennington for functionality of this interaction and suggests a role for this Biomedical Research Center, Baton Rouge, LA) for G  antiserum, Dr. i 3 interaction in the regulation of cell polarity and cell division. John Hildebrandt (Medical University of South Carolina, Charleston, 44 314 The 180-amino acid kinase domain (Lys –Pro ) of LKB1 is SC) for G  antiserum, and Dr. Stephen Graber (University of West i 1 Virginia, Morgantown, WV) for purified G  . We also thank Drs. upstream of the region interacting with AGS3 (Fig. 1). To i 1 Martin and St. Johnston for providing a preprint of Ref. 21 and Dr. Alan determine whether AGS3 is phosphorylated by LKB1, we per- Hall for sharing information cited in Footnote 4. We appreciate the formed in vitro kinase assays using LKB1 immunoprecipitated technical assistance provided by Maureen Fallon and Dallis Green. from LKB1-transfected COS7 cells and purified GST-AGS3 fusion proteins as substrate (Fig. 2A). 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These data suggest Bignell, G., Warren, W., Aminoff, M., Hoglund, P., Jarvinen, H., Kristo, P., that the GPR motif is either itself the site of phosphorylation Pelin, K., Ridanpaa, M., Salovaara, R., Toro, T., Bodmer, W., Olschwang, S., Olsen, A. S., Stratton, M. R., de la Chapelle, A., and Aaltonen, L. A. (1998) and docks within the active site of LKB1 or it is an additional Nature 391, 184 –187 anchor for protein interaction. Peptidomimetics derived from 17. Sapkota, G. P., Kieloch, A., Lizcano, J. M., Lain, S., Arthur, J. S., Williams, the GPR motif may actually be a path for the development of M. R., Morrice, N., Deak, M., and Alessi, D. R. (2001) J. Biol. Chem. 276, 19469 –19482 LKB1 inhibitors. 18. Jishage, K., Nezu, J., Kawase, Y., Iwata, T., Watanabe, M., Miyoshi, A., Ose, As the GPR peptide effectively blocked AGS3-GPR phospho- A., Habu, K., Kake, T., Kamada, N., Ueda, O., Kinoshita, M., Jenne, D. E., Shimane, M., and Suzuki, H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, rylation by LKB1 immunoprecipitates, we asked whether phos- 8903– 8908 phorylation within the GPR motif could potentially influence 19. Watts, J. L., Morton, D. G., Bestman, J., and Kemphues, K. J. (2000) Devel- the interaction of AGS3 with G-proteins. We initially addressed opment 127, 1467–1475 20. Knoblich, J. A. (2001) Nat. Rev. Mol. Cell. Biol. 2, 11–20 this possibility by placing a phosphate moiety on a serine found 21. Martin, S. G., and St. Johnston, D. (2003) Nature 421, 379 –384 in the core of the GPR motif. A 28-amino acid peptide encom- 22. Nezu, J., Oku, A., and Shimane, M. (1999) Biochem. Biophys. Res. Commun. passing the core consensus GPR motif effectively inhibits the 261, 750 –755 23. Kang, S., Liao, P., Gage, D. A., and Esselman, W. J. (1997) J. Biol. Chem. 272, interaction of GPR-containing proteins with G  and also mim- 11588 –11596 ics the action of GPR-containing proteins by inhibiting the 24. Kidd, T., Brose, K., Mitchell, K. J., Fetter, R. D., Tessier-Lavigne, M., Goodman, C. S., and Tear, G. (1998) Cell 92, 205–215 binding of GTPS to G-protein (28). This action of the GPR 25. Bernier, G., Mathieu, M., De Repentigny, Y., Vidal, S. M., and Kothary, R. peptide involves discrete residues within the GPR motif (4, 31), (1996) Genomics 38, 19 –29 mutation of which leads to a loss of activity as indicated for the 26. Umekage, T., and Kato, K. (1991) FEBS Lett. 286, 147–151 27. Smith, D. P., Spicer, J., Smith, A., Swift, S., and Ashworth, A. (1999) Hum. Q22A peptide (28, 31) (Fig. 3A). Phosphorylation of a serine Mol. Genet. 8, 1479 –1485 residue within the GPR motif immediately downstream of the 28. Peterson, Y. K., Bernard, M. L., Ma, H., Hazard, S., III, Graber, S. G., and invariant residue Gln markedly decreased the ability of the Lanier, S. M. (2000) J. Biol. Chem. 275, 33193–33196 29. Tiainen, M., Vaahtomeri, K., Ylikorkala, A., and Makela, T. P. (2002) Hum. GPR motif to interact with G  and to inhibit GTPS binding to Mol. Genet. 11, 1497–1504 G  (Fig. 3, A and B). 30. Du, Q., Stukenberg, P. T., and Macara, I. G. (2001) Nat. Cell Biol. 3, 1069 –1075 LKB1 and other serine/threonine kinases may exert a regu- 31. Peterson, Y. K., Hazard, S., III, Graber, S. G., and Lanier, S. M. (2002) J. Biol. latory influence on the interaction of G-protein  subunits with Chem. 277, 6767– 6770 GPR motif-containing proteins by phosphorylation of residues 32. Hollinger, S., Ramineni, S., and Hepler, J. R. (2003) Biochemistry 42, 811– 819 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Interaction of Activator of G-protein Signaling 3 (AGS3) with LKB1, a Serine/Threonine Kinase Involved in Cell Polarity and Cell Cycle Progression

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 26, Issue of June 27, pp. 23217–23220, 2003 Accelerated Publication © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. where both proteins are involved in cell polarity. LKB1 Interaction of Activator of immunoprecipitates from COS7 cells transfected with G-protein Signaling 3 (AGS3) LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR with LKB1, a Serine/Threonine domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phos- Kinase Involved in Cell Polarity phate moiety within a consensus GPR motif reduced the and Cell Cycle Progression ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may PHOSPHORYLATION OF THE G-PROTEIN be a general mechanism regulating the interaction of REGULATORY (GPR) MOTIF AS A REGULATORY GPR-containing proteins with G-proteins. Such a mech- MECHANISM FOR THE INTERACTION OF GPR anism may be of particular note in regard to localized MOTIFS WITH G * signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regu- Received for publication, December 11, 2002, lated by heterotrimeric G-proteins that occur independ- and in revised form, April 24, 2003 ently of a typical G-protein-coupled receptor. Published, JBC Papers in Press, April 28, 2003, DOI 10.1074/jbc.C200686200 Joe B. Blumer‡§, Michael L. Bernard¶, AGS3 was identified in a functional screen for receptor- Yuri K. Peterson‡, Jun-ichi Nezu**, Peter Chung¶, independent activators of G-protein signaling (1, 2). Surpris- Dara J. Dunican‡‡, Juergen A. Knoblich‡‡, ingly the activation of G-protein signaling in the functional and Stephen M. Lanier‡§§ screen was independent of nucleotide exchange on the G sub- From the **Chugai Pharmaceutical Co., Ltd., Tsukuba unit suggesting unexpected mechanisms for regulating the ac- Research Laboratory, 153-2 Nagai Niihari, Ibaraki tivation state of heterotrimeric G-proteins. AGS3 interacts 300-4101, Japan, the ‡‡Research Institute of Molecular Pathology (IMP), Dr. Bohr Gasse 7, A-1030 Vienna, with G-proteins (G /G ) via its four G-protein regulatory (GPR) i o Austria, the ¶Department of Pharmacology, Medical or GoLoco motifs, each of which interacts with G (G  G ) and i o University of South Carolina, Charleston, South stabilizes the GDP-bound conformation of G (3). The GPR Carolina 29425, and the ‡Department of Pharmacology motif is also found in other proteins including LGN, RGS12, and Experimental Therapeutics, Louisiana State RGS14, Rap1GAPII, Pcp2, and G18.1b (1, 4). AGS3 also contains University Health Sciences Center, New Orleans, seven tetratricopeptide repeats (TPRs) in the first half of the Louisiana 70112 protein, and these domains may serve as a regulatory domain for Activator of G-protein signaling 3 (AGS3) has a modu- the GPR-G-protein interaction, or they may target the protein to lar domain structure consisting of seven tetratricopep- different microdomains within the cell (5). A similar motif struc- tide repeats (TPRs) and four G-protein regulatory (GPR) ture is found in the AGS3-related protein LGN in mammals (6, 7) motifs. Each GPR motif binds to the  subunit of G /G i o as well as in the AGS3/LGN ortholog Pins in Drosophila mela- (G  > G ) stabilizing the GDP-bound conformation of i o nogaster, which is a key determinant of cell polarity (8 –12). A G and apparently competing with G for G bind- GDP role for GPR-containing proteins and G-proteins in cell polarity is ing. As an initial approach to identify regulatory mech- also suggested by studies in Caenorhabditis elegans (13, 14). anisms for AGS3-G-protein interactions, a yeast two-hy- AGS3 and other accessory proteins (proteins distinct from brid screen was initiated using the TPR and linker receptors, G-proteins, and effectors) may influence receptor- region of AGS3 as bait. This screen identified the serine/ mediated signaling events and/or mediate signal input to G- threonine kinase LKB1, which is involved in the regula- tion of cell cycle progression and polarity. Protein inter- proteins independently of a G-protein coupled receptor. Such action assays in mammalian systems using transfected proteins may also serve as alternative binding partners for cells or brain lysate indicated the regulated formation of G-protein subunits independently of heterotrimer formation (1, a protein complex consisting of LKB1, AGS3, and G- 2, 15), and the existence of these accessory proteins suggests proteins. The interaction between AGS3 and LKB1 was unexpected functional roles for G-proteins within the cell. As also observed with orthologous proteins in Drosophila an initial approach to define the cellular control mechanisms for AGS3-G-protein interactions, we sought to identify binding * This work was supported in part by National Institutes of Health partners for the TPR domains of AGS3. Grants MH90531 (to S. M. L.), NS24821 (to S. M. L.), and F32MH65092 We isolated several candidate AGS3-TPR-interacting pro- (to J. B. B.). The costs of publication of this article were defrayed in part teins in a yeast two-hybrid screen, one of which corresponded to by the payment of page charges. This article must therefore be hereby the carboxyl-terminal 107 amino acids of LKB1, also known as marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. serine/threonine kinase 11 (STK11) (16). Loss of LKB1 is im- § Recipient of an individual National Research Service Award sup- plicated in Peutz-Jeghers syndrome, a rare inherited intestinal ported by National Institutes of Health Grant F32MH65092. Recipient of a Medical Scientist Training Program Fellowship sup- ported by National Institutes of Health Grant T32-GM08716. The abbreviations used are: AGS3, activator of G-protein signaling §§ Recipient of a Research Scholar Award from Yamanouchi Phar- 3; TPR, tetratricopeptide repeat; GPR, G-protein regulatory; Pins, Part- maceutical Company, Inc. and the David R. Bethune/Lederle Labora- ner of Inscuteable; Ade, adenine; GST, glutathione S-transferase; tories Professor of Pharmacology. To whom correspondence should be MACF, microtubule/actin cross-linking factor; STK11, serine/threonine addressed: Dept. of Pharmacology, Louisiana State University Health kinase 11; CT, carboxyl terminus; GTPS, guanosine 5-3-O-(thio)tri- Sciences Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504- phosphate; Dm, Drosophila melanogaster; RGS, regulator of G-protein 568-4740; E-mail: [email protected]. signaling; GAP, GTPase-activating protein. This paper is available on line at http://www.jbc.org 23217 This is an Open Access article under the CC BY license. 23218 LKB1 and Phosphorylation of AGS3-GPR Domain polyposis syndrome (16 –18), and LKB1 is actually the mam- malian counterpart of the C. elegans gene par-4, which is a member of a group of polarity-determining genes during em- bryogenesis in both C. elegans and Drosophila (19 –21). Signif- icantly LKB1 phosphorylates AGS3 in its GPR domain, and this was completely blocked by a consensus GPR motif peptide. Place- ment of a phosphate moiety within a consensus GPR motif mark- edly reduced the ability of the peptide to interact with G-proteins suggesting that phosphorylation of GPR motifs may be a general mechanism regulating the interaction of GPR-containing pro- teins with G-proteins. Such a mechanism may be of particular note in regard to the localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur in- dependently of a typical G-protein coupled receptor. EXPERIMENTAL PROCEDURES FIG.1. Interaction of LKB1 with AGS3. A, rat brain (2 mg) lysate was preincubated with 30 M GDP or 30 M GTPS, 25 mM MgCl at Materials—Yeast strains pretransformed with prey libraries, c-Myc 24 °C for 30 min. Lysates were then incubated with 500 nM GST or monoclonal antiserum, and KC-8 chemically competent cells were ob- 330 436 GST-LKB1-CT (Asp –Gln )for1hat24 °C. Protein complexes were tained from Clontech (Palo Alto, CA). Bait vector pGBKT7 and yeast captured by glutathione-Sepharose beads and analyzed by immunoblot- strains Y187 and AH109 were kindly provided by Dr. Tim McQuinn ting following SDS-PAGE. Membrane transfers were first blotted with 32 32 (Medical University of South Carolina). [ - P]ATP and [ P]orthophos- AGS3 antiserum and then stripped and reprobed with G  antiserum. i 3 phate were obtained from PerkinElmer Life Sciences. Sodium or- The Input lane contains ⁄10 of the lysate volume used for each interac- thovanadate and RNase A were obtained from Sigma. Okadaic acid was tion assay. The data are representative of two experiments. B, LKB1 obtained from Calbiochem. pMAL-c2x and amylose-agarose beads were co-immunoprecipitates AGS3. COS7 cells transiently transfected with obtained from New England Biolabs (Boston, MA). Other materials empty vector (V), pcDNA3::AGS3, or pcDNA3::AGS3  pcDNA3::LKB1 were obtained as described elsewhere (3, 7). were lysed in Nonidet P-40 buffer prior to immunoprecipitation with 1 462 Yeast Two-hybrid Screening—AGS3-TPR (Met –Ile ) was gener- LKB1-specific antiserum and immunoblotting with AGS3-specific ated by PCR. Restriction enzyme-digested PCR products were sub- (PEP32) and LKB-specific (P6) antisera. C, Drosophila AGS3 ortholog cloned into pGBKT7 to generate the TPR bait construct. TPR and Pins interacts with Drosophila LKB1 in embryo lysates. Drosophila empty pGBKT7 vector were transformed into AH109 by the lithium embryo lysates were immunoprecipitated with DmLKB1 or Pins anti- acetate method. Expression of bait fusion proteins was confirmed by serum and association of these two proteins was evaluated by SDS- PAGE and immunoblotting for Pins (top panel) and DmLKB1 (bottom immunoblotting with anti-c-Myc. Basal activity of bait strains was panel). The input lane represents ⁄40 of the lysate volume used for each assayed by nutritional selection. AH109 yeast strains expressing TPR immunoprecipitation. IP, immunoprecipitation. as bait were mated with Y187 yeast strains expressing an 11-day-old mouse embryo cDNA library by following the manufacturer’s protocol. The mated yeast culture was plated onto 120 quadruple dropout rabbit anti-Pins (1:100). Mouse anti-Drosophila LKB1 was generated (Trp Leu His Ade ) plates that were then incubated at 30 °C for 7 against a maltose-binding protein-LKB1 fusion protein and recognizes days. -Galactosidase activity was screened using the colony-lift filter a single 75-kDa band in embryonic extracts. Rabbit anti-Pins was assay according to the manufacturer’s directions using diploid p53/ described before (11). SV40 large T antigen interaction (diploid strain PJ69-2A[pVA3-1] Kinase Assay—LKB1 immunoprecipitates were washed three times Y187[pTD1-1]) as a positive control as supplied by the manufacturer. with Nonidet P-40 lysis buffer and three times with kinase buffer A (50 Yeast plasmid DNA was isolated and used to transform competent mM Tris, pH 7.5, 0.1% -mercaptoethanol, 0.1 mM EGTA, 10 mM MnCl , KC-8 Escherichia coli cells. Transformants containing the prey vector 0.5 M okadaic acid) (17). The Gamma-Bind Sepharose was then resus- were selected by plating onto M9 Leu plates. Plasmids isolated from pended in kinase buffer A containing 10 M [- P]ATP (1000 cpm/pmol) KC-8 transformants were transformed into XL1-Blue E. coli cells for and 1 M purified GST fusion protein. Reactions were incubated at further processing and retransformation of yeast strains. 30 °C for 1 h. The Gamma-Bind Sepharose beads were pelleted, and the Immunoprecipitation and Cell Labeling—100-mm dishes of conflu- supernatant was removed and incubated with glutathione-Sepharose ent COS7 cells were transfected with either 10 g of empty vector (Amersham Biosciences) for 30 min at 24 °C to isolate the GST fusion (pcDNA3), 5 g of pcDNA3::AGS3  5 g of empty vector, or 5 gof proteins. The glutathione-Sepharose was pelleted and washed three pcDNA3::AGS3  5 g of pcDNA3::LKB1 (mouse). After 24 h, cells were times with kinase buffer A, resuspended in 5 protein sample buffer, lysed in Nonidet P-40 lysis buffer and incubated on ice for 1 h. The and placed in a boiling water bath for 3 min followed by SDS-PAGE and lysate was centrifuged at 100,000  g for 30 min at 4 °C and precleared autoradiography. with Gamma-Bind Sepharose (Amersham Biosciences). The precleared RESULTS AND DISCUSSION lysates (1 mg of protein) were incubated with 5 g of anti-LKB1 (Up- state Biotechnology, Inc., Lake Placid, NY) for 12–18hat4 °C. Gamma- Although AGS3 clearly interacts with G-proteins and the Bind Sepharose was added, and incubation continued for 30 min. The GPR motifs in AGS3 and other GPR-containing proteins stabi- resin was pelleted and used for kinase assays. Samples were washed lize the GDP-bound conformation of G, only a subpopulation three times with Nonidet P-40 lysis buffer, resuspended in 5 protein of AGS3 and G-proteins are associated with each other in brain sample buffer, and placed in a boiling water bath for 3 min followed by SDS-PAGE and immunoblotting with AGS3-specific (PEP32) (3) and lysates (3), and the two proteins exhibit minimal overlap in LKB-specific (P6) (22) antisera. PC12 cells were labeled with terms of their subcellular distribution (5). These data suggest [ P]orthophosphate (8500 –9120 Ci/mmol) according to Kang et al. (23) that the interaction between AGS3 and G-proteins is a regu- except that phosphate-free Dulbecco’s modified Eagle’s medium was used. lated event. As part of a broader strategy to address this issue For experiments with the Drosophila proteins, 3.5–7-h-old wild-type and define the role of AGS3-G-protein interactions in cellular Drosophila embryos were collected, dechorionated, and lysed by Dounce function, we used a protein interaction screen to identify bind- homogenization in ice-cold embryo lysis buffer (25 mM Tris-HCl, pH 8.0, 27.5 mM NaCl, 20 mM KCl, 25 mM sucrose, 10 mM EDTA, 10 mM EGTA, ing partners for the TPR and linker region of AGS3. A yeast 1mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10% glycerol, two-hybrid screen of a mouse 11-day-old embryonic cDNA li- 0.1% Nonidet P-40 supplemented with protease and phosphatase inhib- 1 462 brary using AGS3-TPR (Met –Ile ) as bait yielded several itors). The resultant lysate was centrifuged at 14,000 rpm in a 4 °C candidate AGS3 binding partners. The screen was run with bench-top centrifuge, and the supernatants were used for immunopre- high stringency by directly using quadruple dropout selection cipitation. Each immunoprecipitation (1:60 dilution of antisera) used (Trp Leu His Ade ) followed by a secondary selection for col- lysate corresponding to 150 l of packed embryos in a total volume of 300 l. Immunoblots were performed with mouse anti-LKB1 (1:200) or onies that exhibited strong -galactosidase activity within 30 LKB1 and Phosphorylation of AGS3-GPR Domain 23219 FIG.3. Influence of GPR phosphorylation on G-protein inter- action. A, protein interaction assays were performed as described under “Experimental Procedures” using 75 nM G  and 300 nM GST- i 1 AGS3 in the presence of 10 M GDP. Peptide concentration  10 M. Similar results were obtained in three separate experiments. The Input lane represents ⁄10 of the total volume in each interaction assay. B, GTP S (500 nM) binding to G  (100 nM) was measured after incuba- tion with increasing concentrations of control GPR consensus peptide and a phosphorylated Ser (PhosphoS16) GPR peptide as described under “Experimental Procedures.” Protein interaction assays and GTPS binding assays were performed as described previously (28). Data are expressed as the percentage of specific binding (0.5 pmol) FIG.2. LKB1 phosphorylates the GPR domain of AGS3. A, top observed in the absence of added peptide and are expressed as the mean  S.E. of two experiments with duplicate determinations. panel, COS7 cells were transfected with 10 g of pcDNA3::LKB1 or empty vector for 24 h prior to immunoprecipitation and immunoblotting with LKB1 antiserum. Bottom panel, purified GST fusion proteins (1 The cDNA clone encoding LKB1 contained the last 107 g/lane) were subjected to SDS-PAGE and Coomassie Blue staining. amino acids of the coding region of LKB1. The interaction of Right panel, LKB1 immunoprecipitates from LKB1-transfected COS7 1 462 1 462 AGS3-TPR (Met –Ile ) with LKB1 in the yeast two-hybrid cells were incubated with 1 M GST, GST-AGS3-TPR (Met –Ile ), or 338 462 463 650 32 GST-AGS3-GPR (Pro –Ser ) in the presence of [- P]ATP for1hat screen required amino acids Asp –Ile in the AGS3 coding 30 °C as described under “Experimental Procedures.” GST fusion pro- region that connects the TPR and GPR domains. Additional teins were purified with a glutathione resin prior to SDS-PAGE and regions of AGS3 and LKB1 may also interact with each other in autoradiography. The data are representative of three experiments. B, 3,4 the context of the full-length proteins. We then asked whether LKB1 kinase assays were performed as described under “Experimental 463 650 Procedures” with GST or GST-AGS3-GPR (Pro –Ser ) fusion pro- the interaction between the carboxyl terminus of LKB1 and teins in the presence or absence of a consensus GPR peptide (TMGEED- AGS3 was observed in a mammalian system using a GST fusion FFDLLAKSQSKRMDDQRVDLAG) or a scrambled control peptide protein of the LKB1 fragment isolated in the yeast two-hybrid (TMGDDQRLLAKSQSKRMEEDFFDVDLAG) at a final concentration 330 436 screen. LKB1-CT (Asp –Gln ) effectively interacted with en- of 100 M. GST fusion proteins were purified with glutathione-Sepha- dogenous, full-length AGS3 in rat brain lysates (Fig. 1). Interest- rose, washed, and subjected to SDS-PAGE and autoradiography. Data are representative of two experiments. C, PC12 cells, which express ingly this complex also contained G  subunits, which is likely endogenous AGS3, were labeled with [ P]orthophosphate 3 h prior to due to an interaction of G-proteins with the GPR motifs of AGS3 cell lysis (500 g of protein) and immunoprecipitated with AGS3 (3). The presence of G-proteins in this complex was nucleotide- antiserum followed by SDS-PAGE and autoradiography. Parallel unla- dependent in that it was not observed in the presence of the beled samples (500 g of protein) were used for immunoblotting with anti-AGS3 antiserum to confirm expression and immunoprecipitation. nonhydrolyzable GTP analog GTPS, which is consistent with Data are representative of two experiments. IP, immunoprecipitation; the demonstrated preference of GPR motifs for the GDP-bound IB, immunoblotting; autorad, autoradiography. conformation of G (1, 3, 28). The interaction of AGS3 itself with LKB1 was not influenced by guanine nucleotides. min. Sixteen cDNAs were isolated, five of which encoded DNA- The LKB1-AGS3 interaction was further addressed with the binding proteins or proteins involved in regulation of transcrip- full-length proteins in the intact cell. Co-transfection of cDNAs tion or translation. Of the remaining 11, one cDNA clone en- encoding full-length AGS3 and LKB1 in COS7 cells and sub- coded an extracellular protein, and five encoded previously sequent immunoprecipitation with LKB1 antiserum resulted unidentified proteins or proteins of unknown function. The in co-immunoprecipitation of AGS3 (Fig. 1B). This interaction remaining cDNA clones encoded portions of murine robo-1, an was specific for LKB1 as immunoprecipitation with LKB1 an- axonal guidance receptor during central nervous system devel- tiserum from cells transfected with AGS3 alone did not co- opment (24); microtubule/actin cross-linking factor (MACF, immunoprecipitate AGS3 (Fig. 1B). In contrast to the results also known as ACF7), a member of the plakin family implicated 330 436 obtained with the GST-LKB1-CT (Asp –Gln ) fusion pro- in epithelial and neuronal polarity (25); MARCKS (myristo- tein in which G  was brought down with the LKB1-CTAGS3 ylated alanine-rich C kinase substrate)-like protein, a regula- complex from brain lysates, G  was not found in the co-immu- tor of actin dynamics, migration, and neuronal development noprecipitation complex of the full-length proteins suggesting (26); and LKB1/STK11 (27). LKB1 and the Drosophila AGS3/ that LKB1 may process incoming signals to regulate the inter- LGN ortholog Pins are both involved in various aspects of cell action between AGS3 and G-proteins or target the protein to a polarity and development. As a tertiary screen to select for microdomain where G-proteins are inaccessible. proteins of potential interest, we asked whether GST fusion To provide further evidence for a functional interaction be- proteins of each of these cDNA clones interacted with full- tween LKB1 and AGS3, we asked whether the interaction was length AGS3 in brain lysates. Only GST-MACF and GST-LKB1 evolutionarily conserved in Drosophila. Drosophila LKB1 effectively pulled down AGS3 from rat brain lysates. We first (DmLKB1) co-immunoprecipitated with the AGS3 ortholog focused our effort on LKB1 as the LKB1 ortholog in C. elegans Pins, and conversely Pins co-immunoprecipitated with was previously identified as a PAR (partitioning defective) gene DmLKB1 in Drosophila embryo lysates, indicating an interac- (19) involved in asymmetric division of C. elegans embryos. AGS3/LGN orthologs or proteins containing GPR motifs are also J. B. Blumer, Y. K. Peterson, and S. M. Lanier, unpublished involved in similar events in Drosophila and C. elegans. observations. AGS3 was also isolated in a yeast two-hybrid screen using LKB1 as M. L. Bernard, J. B. Blumer, and S. M. Lanier, unpublished bait, and both proteins were reported to be involved in astrocyte polar- observations. ity (A. Hall, personal communication). 23220 LKB1 and Phosphorylation of AGS3-GPR Domain tion of the full-length Drosophila proteins (Fig. 1C). In both the within the GPR motif. GPR motifs are found in several proteins COS7 transfectants and the Drosophila embryos only a sub- involved in signal propagation including LGN, RGS12, RGS14, population of AGS3 (or Pins) was actually complexed with Rap1GAP, Pcp2, and G18.1b (1, 4). A recent report also sug- LKB1 following immunoprecipitation. This may reflect the af- gested that phosphorylation near the GPR motif of RGS14 may finity of the interaction, stoichiometric considerations, and/or influence GPR-G  interaction (32). Such a mechanism may regulation of the interaction by an as yet undefined signal(s). The allow the discrete regulation of G-protein signaling in specific AGS3 ortholog in Drosophila plays a critical role in cell polarity subcellular compartments that occurs independently of a G- that apparently also involves heterotrimeric G-proteins (8 –12). protein-coupled receptor. The localized regulation of such The LKB1 ortholog in Drosophila was also recently identified in events is a signature mechanism for the determination of cell a genetic screen for defects in oocyte and epithelial cell polarity polarity and asymmetric cell division observed in stem cells (21). The demonstration of an interaction between LKB1 and during tissue development. AGS3/LGN orthologs in Drosophila provides additional evidence Acknowledgments—We thank Dr. Thomas W. Gettys (Pennington for functionality of this interaction and suggests a role for this Biomedical Research Center, Baton Rouge, LA) for G  antiserum, Dr. i 3 interaction in the regulation of cell polarity and cell division. John Hildebrandt (Medical University of South Carolina, Charleston, 44 314 The 180-amino acid kinase domain (Lys –Pro ) of LKB1 is SC) for G  antiserum, and Dr. Stephen Graber (University of West i 1 Virginia, Morgantown, WV) for purified G  . We also thank Drs. upstream of the region interacting with AGS3 (Fig. 1). To i 1 Martin and St. Johnston for providing a preprint of Ref. 21 and Dr. Alan determine whether AGS3 is phosphorylated by LKB1, we per- Hall for sharing information cited in Footnote 4. We appreciate the formed in vitro kinase assays using LKB1 immunoprecipitated technical assistance provided by Maureen Fallon and Dallis Green. from LKB1-transfected COS7 cells and purified GST-AGS3 fusion proteins as substrate (Fig. 2A). 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Published: Jun 1, 2003

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