Sam68 is a member of a growing family of RNA-binding proteins that contains an extended K homology (KH) domain embedded in a larger domain called the GSG (GRP33,Sam68, GLD1) domain. To identify GSG domain family members, we searched data bases for expressed sequence tags encoding related portions of the Sam68 KH domain. Here we report the identification of two novel Drosophila KH domain proteins, which we termed KEP1 (KH encompassingprotein) and SAM. SAM bears sequence identity with mammalian Sam68 and may be the Drosophila Sam68 homolog. We demonstrate that SAM, KEP1, and the recently identifiedDrosophila Who/How are RNA-binding proteins that are able to self-associate into homomultimers. The GSG domain of KEP1 and SAM was necessary to mediate the RNA binding and self-association. To elucidate the cellular roles of these proteins, SAM, KEP1, and Who/How were expressed in mammalian and Drosophila S2 cells. KEP1 and Who/How were nuclear and SAM was cytoplasmic. The expression of KEP1 and SAM, but not Who/How, activated apoptotic pathways in Drosophila S2 cells. The identification of KEP1 and SAM implies that a large GSG domain protein family exists and helps redefine the boundaries of the GSG domain. Taken together, our data suggest that KEP1 and SAM may play a role in the activation or regulation of apoptosis and further implicate the GSG domain in RNA binding and oligomerization.
The Mpv 20 transgenic mouse strain was created by infection of embryos with a defective retrovirus. When Mpv 20 heterozygous animals were crossed, no homozygous neonatal mice or midgestation embryos were identified. When embryos from heterozygous crosses were cultured in vitro, approximately one quarter arrested as uncompacted eight-cell embryos, indicating that proviral insertion resulted in a recessive lethal defect whose phenotype was manifest very early in development. Molecular cloning of the Mpv 20 insertion site revealed that the provirus had disrupted the Npat gene, a gene of unknown function, resulting in the production of a truncated Npat mRNA. Expression of the closely linked Atm gene was found to be unaffected by the provirus.
Dual leucine zipper-bearing kinase (DLK) is a mixed-lineage kinase family member that acts as an upstream activator of the c-Jun N-terminal kinases. As opposed to other components of this pathway, very little is currently known regarding the mechanisms by which DLK is regulated in mammalian cells. Here we identify the stress-inducible heat shock protein 70 (Hsp70) as a negative regulator of DLK expression and activity. Support for this notion derives from data showing that Hsp70 induces the proteasomal degradation of DLK when both proteins are co-expressed in COS-7 cells. Hsp70-mediated degradation occurs with expression of wild-type DLK, which functions as a constitutively activated protein in these cells but not kinase-defective DLK. Interestingly, the Hsp70 co-chaperone CHIP, an E3 ubiquitin ligase, seems to be indispensable for this process since Hsp70 failed to induce DLK degradation in COS-7 cells expressing a CHIP mutant unable to catalyze ubiquitination or in immortalized fibroblasts derived from CHIP knock-out mice. Consistent with these data, we have found that endogenous DLK becomes sensitive to CHIP-dependent proteasomal degradation when it is activated by okadaic acid and that down-regulation of Hsp70 levels with an Hsp70 antisense attenuates this sensitivity. Therefore, our studies suggest that Hsp70 contributes to the regulation of activated DLK by promoting its CHIP-dependent proteasomal degradation. Dual leucine zipper-bearing kinase (DLK) is a mixed-lineage kinase family member that acts as an upstream activator of the c-Jun N-terminal kinases. As opposed to other components of this pathway, very little is currently known regarding the mechanisms by which DLK is regulated in mammalian cells. Here we identify the stress-inducible heat shock protein 70 (Hsp70) as a negative regulator of DLK expression and activity. Support for this notion derives from data showing that Hsp70 induces the proteasomal degradation of DLK when both proteins are co-expressed in COS-7 cells. Hsp70-mediated degradation occurs with expression of wild-type DLK, which functions as a constitutively activated protein in these cells but not kinase-defective DLK. Interestingly, the Hsp70 co-chaperone CHIP, an E3 ubiquitin ligase, seems to be indispensable for this process since Hsp70 failed to induce DLK degradation in COS-7 cells expressing a CHIP mutant unable to catalyze ubiquitination or in immortalized fibroblasts derived from CHIP knock-out mice. Consistent with these data, we have found that endogenous DLK becomes sensitive to CHIP-dependent proteasomal degradation when it is activated by okadaic acid and that down-regulation of Hsp70 levels with an Hsp70 antisense attenuates this sensitivity. Therefore, our studies suggest that Hsp70 contributes to the regulation of activated DLK by promoting its CHIP-dependent proteasomal degradation. Dual leucine zipper-bearing kinase (DLK) 2The abbreviations used are: DLK, dual leucine zipper-bearing kinase; MLK, mixed-lineage kinase; JNK, c-Jun N-terminal kinase; Hsp70, stress-inducible heat shock protein 70; CHIP, C terminus of Hsc70-interacting protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PMSF, phenylmethylsulfonyl fluoride. 2The abbreviations used are: DLK, dual leucine zipper-bearing kinase; MLK, mixed-lineage kinase; JNK, c-Jun N-terminal kinase; Hsp70, stress-inducible heat shock protein 70; CHIP, C terminus of Hsc70-interacting protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PMSF, phenylmethylsulfonyl fluoride. is a serine/threonine kinase that belongs to a family of mitogen-activated protein kinase kinase kinases, known as mixed-lineage kinases (MLKs) (1Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell Biol. 2002; 3: 663-672Crossref PubMed Scopus (455) Google Scholar). Members of this family, which also include MLK1, MLK2, MLK3, MLK4, leucine zipper-bearing kinase, and leucine zipper and sterile α-motif kinase (1Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell Biol. 2002; 3: 663-672Crossref PubMed Scopus (455) Google Scholar), are characterized at the structural level by the presence of a catalytic domain bearing amino acid motifs found in serine/threonine and tyrosine kinases and one or two leucine zipper motifs, which regulate their activity by mediating protein dimerization or oligomerization (2Ezoe K. Lee S.T. Strunk K.M. Spritz R.A. Oncogene. 1994; 9: 935-938PubMed Google Scholar, 3Gallo K.A. Mark M.R. Scadden D.T. Wang Z. Gu Q. Godowski P.J. J. Biol. Chem. 1994; 269: 15092-15100Abstract Full Text PDF PubMed Google Scholar, 4Hirai S. Katoh M. Terada M. Kyriakis J.M. Zon L.I. Rana A. Avruch J. Ohno S. J. Biol. Chem. 1997; 272: 15167-15173Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 5Sakuma H. Ikeda A. Oka S. Kozutsumi Y. Zanetta J.P. Kawasaki T. J. Biol. Chem. 1997; 272: 28622-28629Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A number of other interesting motifs that are likely important for protein binding have also been identified in specific members of the MLK family. For instance, MLK2 and MLK3 contain a Src homology 3 (SH3) domain in their N-terminal region that binds, respectively, the GTPase dynamin and the Ste20-related protein kinase HPK1 (6Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (199) Google Scholar, 7Rasmussen R.K. Rusak J. Price G. Robinson P.J. Simpson R.J. Dorow D.S. Biochem. J. 1998; 335: 119-124Crossref PubMed Scopus (23) Google Scholar). Both MLK proteins also possess a functional Cdc42/Rac interactive binding (CRIB) motif that mediates association with Cdc42 and Rac1 in a GTP-dependent manner (8Nagata K. Puls A. Futter C. Aspenstrom P. Schaefer E. Nakata T. Hirokawa N. Hall A. EMBO J. 1998; 17: 149-158Crossref PubMed Scopus (233) Google Scholar, 9Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). The importance of the MLKs as signaling molecules is highlighted by the fact that these proteins act as key regulators of the c-Jun N-terminal kinase (JNK) subgroup of mitogen-activated protein kinases (1Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell Biol. 2002; 3: 663-672Crossref PubMed Scopus (455) Google Scholar). Specifically, all MLK family members regulate the JNK pathway by phosphorylating and activating the JNK direct upstream activators MKK4 and MKK7 (10Cuenda A. Dorow D.S. Biochem. J. 1998; 333: 11-15Crossref PubMed Scopus (57) Google Scholar, 11Hirai S. Noda K. Moriguchi T. Nishida E. Yamashita A. Deyama T. Fukuyama K. Ohno S. J. Biol. Chem. 1998; 273: 7406-7412Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 12Merritt S.E. Mata M. Nihalani D. Zhu C. Hu X. Holzman L.B. J. Biol. Chem. 1999; 274: 10195-10202Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 13Rana A. Gallo K. Godowski P. Hirai S.-i. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 14Tibbles L.A. Ing Y.L. Kiefer F. Chan J. Iscove N. Woodgett J.R. Lassam N.J. EMBO J. 1996; 15: 7026-7035Crossref PubMed Scopus (280) Google Scholar). In addition to their role in catalyzing JNK activation, MLKs are also known to contribute to apoptosis in neuronal cells. Indeed, when dominant negative forms of MLKs were expressed in neuronal PC12 cells and sympathetic neurons, death caused by nerve growth factor deprivation was severely inhibited (15Xu Z. Maroney A.C. Dobrzanski P. Kukekov N.V. Greene L.A. Mol. Cell. Biol. 2001; 21: 4713-4724Crossref PubMed Scopus (223) Google Scholar). Evidence supporting the involvement of the MLKs in apoptosis also derives from studies with the MLK inhibitor CEP1347 (16Maroney A.C. Finn J.P. Connors T.J. Durkin J.T. Angeles T. Gressner G. Xu Z. Meyer S.L. Savage M.J. Greene L.A. Scott R.W. Vaught J.L. J. Biol. Chem. 2001; 276: 25302-25308Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), which provides neuroprotection against numerous death-inducing stimuli (17Maroney A.C. Glicksman M.A. Basma A.N. Walton K.M. Knight Jr., E. Murphy C.A. Bartlett B.A. Finn J.P. Angeles T. Matsuda Y. Neff N.T. Dionne C.A. J. Neurosci. 1998; 18: 104-111Crossref PubMed Google Scholar, 18Maroney A.C. Finn J.P. Bozyczko-Coyne D. O'Kane T.M. Neff N.T. Tolkovsky A.M. Park D.S. Yan C.Y. Troy C.M. Greene L.A. J. Neurochem. 1999; 73: 1901-1912PubMed Google Scholar, 19Saporito M.S. Hudkins R.L. Maroney A.C. Prog. Med. Chem. 2002; 40: 23-62Crossref PubMed Scopus (94) Google Scholar). As is the case for the majority of MLK family members, relatively little is known about the mechanisms responsible for the activation and regulation of DLK in mammalian cells. Current evidence suggests that the leucine zipper domain of DLK plays a role in the activation process, as deletion of this region prevents dimerization, autophosphorylation, and stimulation of the JNK pathway (20Nihalani D. Merritt S. Holzman L.B. J. Biol. Chem. 2000; 275: 7273-7279Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Work from a number of laboratories has also established that the regulation of DLK involves heterologous interactions with scaffolding and inhibitory proteins. The binding of DLK to these proteins, in particular JNK-interacting protein (JIP)-1 and MAPK upstream kinase (MUK)-binding inhibitory protein (MBIP), is likely to play an important role in DLK regulation by preventing its dimerization (21Fukuyama K. Yoshida M. Yamashita A. Deyama T. Baba M. Suzuki A. Mohri H. Ikezawa Z. Nakajima H. Hirai S. Ohno S. J. Biol. Chem. 2000; 275: 21247-21254Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Nihalani D. Meyer D. Pajni S. Holzman L.B. EMBO J. 2001; 20: 3447-3458Crossref PubMed Scopus (87) Google Scholar, 23Nihalani D. Wong H.N. Holzman L.B. J. Biol. Chem. 2003; 278: 28694-28702Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Another important mechanism by which DLK is proposed to be regulated is through changes in its phosphorylation status. Experimental evidence supporting the involvement of phosphorylation in DLK regulation derives from the observation that oligomerization-dependent autophosphorylation is required for activation of DLK and stimulation of the JNK pathway (20Nihalani D. Merritt S. Holzman L.B. J. Biol. Chem. 2000; 275: 7273-7279Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Furthermore, other studies have revealed that DLK exists in part as a phosphoprotein in vivo and that treatment of cells with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, results in accumulation of phosphorylated DLK (24Mata M. Merritt S.E. Fan G. Yu G.G. Holzman L.B. J. Biol. Chem. 1996; 271: 16888-16896Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Finally, Nakata et al. (25Nakata K. Abrams B. Grill B. Goncharov A. Huang X. Chisholm A.D. Jin Y. Cell. 2005; 120: 407-420Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) recently demonstrated that DLK protein levels in Caenorhabditis elegans are down-regulated by an E3 ubiquitin ligase, termed RPM-1, which was found to stimulate DLK ubiquitination. In the current study we report that activated DLK either exogenously or endogenously expressed is down-regulated by the stress-inducible heat shock protein 70 (Hsp70), a molecular chaperone that confers cytoprotection in response to various stress stimuli (26Beere H.M. J. Cell Sci. 2004; 117: 2641-2651Crossref PubMed Scopus (509) Google Scholar, 27Garrido C. Schmitt E. Cande´ C. Vahsen N. Parcellier A. Kroemer G. Cell Cycle. 2003; 2: 579-584Crossref PubMed Scopus (202) Google Scholar, 28Kiang J.G. Tsokos G.C. Pharmacol. Ther. 1998; 80: 183-201Crossref PubMed Scopus (997) Google Scholar, 29Takayama S. Reed J.C. Homma S. Oncogene. 2003; 22: 9041-9047Crossref PubMed Scopus (400) Google Scholar). Our study also shows that Hsp70-dependent DLK down-regulation is mediated by a proteasome-dependent mechanism involving the CHIP ubiquitin ligase. Thus, our results support a role for Hsp70 and CHIP as important regulators of DLK protein levels. Chemicals, Reagents, and Antibodies—The proteasome inhibitor lactacystin and the rabbit polyclonal antibody against actin were purchased from Sigma-Aldrich. The mouse monoclonal antibody for the detection of phospho-JNK and the rabbit polyclonal antibody insensitive to the phosphorylation state of JNK were purchased from Cell Signaling Technology Inc. (Beverly, MA). The mouse monoclonal antibodies against the T7 and the hexahistidine tag sequences were from Novagen, Inc. (Madison, WI) and Invitrogen, respectively. The anti-Myc monoclonal antibody (clone 9E10) and okadaic acid were obtained from Calbiochem-Novabiochem. The rabbit polyclonal antibodies against DLK, Hsp70, Hsp27, Hsp40, Hsp60, Hsc70, Hsp90α, Hsp90β, and CHIP were described previously (30Douziech M. Laberge G. Grondin G. Daigle N. Blouin R. J. Histochem. Cytochem. 1999; 47: 1287-1296Crossref PubMed Scopus (16) Google Scholar, 31Tanguay R.M. Wu Y. Khandjian E.W. Dev. Genet. 1993; 14: 112-118Crossref PubMed Scopus (141) Google Scholar, 32Laplante A.F. Moulin V. Auger F.A. Landry J. Morrow G. Tanguay R.M. Germain L. J. Histochem. Cytochem. 1998; 48: 1291-1301Crossref Scopus (151) Google Scholar, 33Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.T. Yin L.-Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (750) Google Scholar). The rabbit anti-ubiquitin antibody was purchased from Rockland Immunochemicals, Inc. (Gilbertsville, PA). Recombinant Hsp70 was purchased from Stressgen Biotechnologies Corp. (Victoria, BC, Canada). Cell culture reagents were from Invitrogen, HyClone Laboratories (Logan, UT), Cambrex Corp. (East Rutherford, NJ), or BIOSOURCE International Inc. (Camarillo, CA). Cell Culture—COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml amphotericin B. Transformed lung fibroblasts derived from CHIP+/+ and CHIP–/– mice were also maintained in DMEM plus 10% FBS. Plasmids and Transfection—The T7-tagged DLK expression vector was generously provided by Dr. S. Hirai (Yokohama City University School of Medicine). The expression vector for T7-tagged catalytically inactive DLK was produced by replacing the invariant lysine at position 185 within kinase subdomain II with arginine using the Stratagene QuikChange site-directed mutagenesis kit (La Jolla, CA). The entire coding region of the cloned gene for human hsp70 (34Hunt C. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6455-6459Crossref PubMed Scopus (700) Google Scholar) was inserted into a mammalian expression vector under the control of the cytomegalovirus promoter (CMV5). A mutant form of Hsp70 lacking the ATP binding domain (Hsp70ΔABD) was constructed with the Hsp70 expression vector by in-frame deletion of amino acid residues 120–428 of the published human Hsp70 sequence (34Hunt C. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6455-6459Crossref PubMed Scopus (700) Google Scholar). pCW8 (expressing Myc-tagged ubiquitin K48R) was a gift from Dr. R. Kopito (35Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1130) Google Scholar). The pcDNA3 expression plasmids encoding wild-type Myc-tagged CHIP (Myc-CHIP) and its U-box deletion mutant (Myc-CHIPΔE4) have been described previously (36Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L. Ho¨hfeld J. Patterson C. Nat. Cell Biol. 2001; 18: 93-96Crossref Scopus (822) Google Scholar). The antisense hsp70 pcDNA3 plasmid, containing a 500-base pair fragment of the human inducible hsp70 cDNA in the antisense orientation (974–475), was a kind gift of Dr. M. Ja¨a¨ttela (37Ja¨a¨ttela¨ M. Wissing D. Kokholm K. Kallunki T. Egeblad M. EMBO J. 1998; 17: 6124-6134Crossref PubMed Scopus (616) Google Scholar). For gene transfer experiments, COS-7, CHIP+/+, and CHIP–/– cells in exponential growth phase were seeded at 2.5 × 105 viable cells/60-mm dish and allowed to recover for 24 h. Thereafter, cells were transfected or cotransfected with the different expression vectors mentioned above using FuGENE 6 transfection reagent (Roche Diagnostics). Cells were harvested and processed for immunoblot analysis 48 h after transfection. When indicated, medium was removed 48 h after transfection, and cells were treated with lactacystin (10 μm) and/or okadaic acid (400 nm) in 3 ml of DMEM supplemented with 10% (v/v) heat-inactivated FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml amphotericin B/60 mm at 37 °C. Then cells were harvested and processed for further analyses. Preparation of Cell Lysates and Immunoblotting—Cells were lysed for 30 min at 4 °C in 15 mm Tris-HCl, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 150 mm NaCl, 1 mm EGTA, 1 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml leupeptin, and 1 μg/ml aprotinin. Lysates were clarified by centrifugation (13,000 rpm for 10 min at 4 °C), and the concentration of total protein in the supernatant fraction was quantified by the modified Bradford protein assay (Bio-Rad). For immunoblotting, equal amounts of proteins were fractionated on 10% reducing SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Roche Diagnostics) using a semidry transfer apparatus (Hoefer Scientific Instruments). Membranes were blocked overnight at 4 °C in 20 mm Tris, pH 7.5, 150 mm NaCl, 0.1% Tween 20 containing 5% skim milk powder before incubation with the primary antibody for 1 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence using secondary horseradish peroxidase-conjugated antibodies (ECL Plus Western blotting kit, Amersham Biosciences). Immunoprecipitation—Cells were lysed for 30 min at 4 °C in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 50 mm NaF, 0.2 mm Na3VO4, 1 mm PMSF, 1 μg/ml leupeptin, and 1 μg/ml aprotinin. Lysates were clarified by centrifugation (13,000 rpm for 10 min at 4 °C), and the concentration of total protein in the supernatant fraction was quantified using the modified Bradford protein assay (Bio-Rad). Typically, 500 μg of protein extracts were incubated for 4 h at 4°C with constant rotation with the primary antibody and protein A-agarose beads. Immune complexes were collected by centrifugation (13,000 rpm for 1 min) and washed three times with lysis buffer. The resulting pellet was resuspended in 2× SDS-PAGE sample buffer, boiled for 5 min, fractionated on 10% SDS-PAGE, and processed for immunoblot analysis as described above. In Vitro Interaction Assay—The full-length coding sequence of wild-type DLK was inserted in-frame with a His-tag sequence in the pTriEx-4 expression vector (Novagen) for production of recombinant proteins using the TNT® T7 Coupled Reticulocyte Lysate system (Promega Corp., Madison, WI). After nonradioactive translation His-tagged wild-type DLK was incubated for 2 h at 30°C alone or in combination with recombinant Hsp70 (1 μg) in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 1 mm PMSF, 1 μg/ml leupeptin, and 1 μg/ml aprotinin. Proteins were then subjected to immunoprecipitation with the anti-Hsp70 antibody, and the resulting immunocomplexes were analyzed by immunoblotting with either the anti-Hsp70 or anti-His antibody. Pulse-Chase Analysis—COS-7 cells transiently expressing T7-tagged wild-type DLK in the presence or absence of Hsp70 were starved in DMEM plus 0.5% FBS without methionine and cysteine for 16 h and then metabolically labeled with [35S]methionine/cysteine (Easy Tag™ EXPRESS, PerkinElmer Life Sciences) for 45 min. Subsequently, cells were chased in nonradioactive medium for the times indicated, lysed, and subjected to immunoprecipitation with the anti-T7 antibody. Immunocomplexes were resolved by SDS-PAGE and visualized by autoradiography. Immunocomplex Kinase Assay for DLK—Cultures of CHIP+/+ and CHIP–/– cells were incubated in the absence or presence of okadaic acid (400 nm) and lactacystin (10 μm) before homogenization in lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 50 mm NaF, 0.2 mm Na3VO4, 1mm PMSF, 1 μg/ml leupeptin, and 1 μg/ml aprotinin). The lysates were clarified by centrifugation, and the concentration of total protein in the supernatant fraction was quantified using the modified Bradford protein assay (Bio-Rad). Typically, 600 μg of protein extract were incubated for 2 h at 4 °C with constant rotation using a polyclonal antibody against DLK (30Douziech M. Laberge G. Grondin G. Daigle N. Blouin R. J. Histochem. Cytochem. 1999; 47: 1287-1296Crossref PubMed Scopus (16) Google Scholar) and protein A-Sepharose beads. After incubation, the immunocomplexes were washed 3 times with lysis buffer and 3 times with kinase buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10 mm MgCl2, 0.5 mm dithiothreitol, 0.1 mm PMSF, 0.2 mm sodium orthovanadate, 1 μg/ml leupeptin, and 1 μg/ml aprotinin). Immunocomplex kinase assays were performed by incubating the immune complexes in 40 μl of kinase buffer containing 2.5 μCi of [γ-32P]ATP (Amersham Biosciences), 25 μm ATP, and 1 μg of myelin basic protein as a substrate. After a 20-min incubation at 30 °C, the reaction was stopped by adding an appropriate volume of 6× SDS-PAGE sample buffer and boiling for 5 min. Phosphorylated proteins were visualized by autoradiography after fractionation by SDS-PAGE. Down-regulation of DLK by Hsp70—During the course of a study recently conducted in our laboratory, we noticed that overexpression of wild-type DLK in MCF-7 human breast cancer cells resulted in the accumulation of a protein with a molecular weight (Mr) of ∼70,000 (p70), as revealed by Coomassie Blue staining of cell extracts fractionated on reducing SDS-PAGE. This protein band was excised from the gel and analyzed by tandem mass spectrometry at the Eastern Quebec Proteomic Center (Universite´ Laval, Que´bec, Canada). A correlative search of the NCBI Protein Data base with the peptide product ion spectra generated by tandem mass spectrometry identified p70 as the stress-inducible protein Hsp70, a molecular chaperone that has been shown to protect cells against the potentially fatal consequences of diverse physiological and environmental insults (26Beere H.M. J. Cell Sci. 2004; 117: 2641-2651Crossref PubMed Scopus (509) Google Scholar, 27Garrido C. Schmitt E. Cande´ C. Vahsen N. Parcellier A. Kroemer G. Cell Cycle. 2003; 2: 579-584Crossref PubMed Scopus (202) Google Scholar). This result was subsequently confirmed by immunoblot analysis of total proteins isolated from MCF-7 cells overexpressing DLK with an antibody specific for the inducible Hsp70 protein (31Tanguay R.M. Wu Y. Khandjian E.W. Dev. Genet. 1993; 14: 112-118Crossref PubMed Scopus (141) Google Scholar). To determine whether DLK overexpression could elicit a similar induction of Hsp70 in another cell system, we transfected COS-7 cells with an expression vector encoding a T7 epitope-tagged form of wild-type DLK. At 48 h after transfection cells were lysed and processed for immunoblot analyses using either anti-T7 or anti-Hsp70 antibodies. The data presented in Fig. 1A demonstrate that overexpressed DLK, which functions as a constitutively activated protein able to induce phosphorylation of endogenous JNK, substantially increased the levels of endogenous Hsp70 when compared with cells transfected with an empty vector. Interestingly, this effect is specific for Hsp70, because expression of the other Hsps examined, including Hsp27, Hsp40, Hsp60, Hsc70, Hsp90α, and Hsp90β, was not changed in DLK-transfected cells. Moreover, although it was expressed at levels comparable with wild-type DLK, a catalytically inactive T7-tagged DLK mutant did not influence the expression of any Hsps in transfected cells. Thus, these results confirm that the ectopic expression of DLK in COS-7 cells is sufficient to up-regulate in a specific manner Hsp70 protein levels and suggest that DLK kinase activity is required for Hsp70 induction. To investigate the physiological relevance of Hsp70 induction in DLK-transfected cells, we next assayed the effects of this molecular chaperone on the steady-state levels of wild-type and kinase-defective T7-DLK in COS-7 cells co-transfected with an expression plasmid for human Hsp70. After transfection, cells were lysed and processed for immunoblot analysis with anti-T7 and anti-Hsp70 antibodies. Results shown in Fig. 1B reveals that Hsp70 co-expression, as demonstrated by the immunoblot data, reduces by ∼80% the abundance of wild-type T7-DLK in transfected cells. In contrast, the levels of the catalytically inactive form of T7-DLK remained constant in response to Hsp70 overexpression. These data indicate that wild-type T7-DLK undergoes down-regulation mediated at least in part by its own activity in cells which overexpressed Hsp70. Because Hsp70 is composed of two functionally distinct domains, an N-terminal ATPase domain responsible for its protein folding function and a C-terminal peptide-binding domain that specifically binds both unfolded and folded substrates (28Kiang J.G. Tsokos G.C. Pharmacol. Ther. 1998; 80: 183-201Crossref PubMed Scopus (997) Google Scholar, 38Milarski K.L. Morimoto R.I. J. Cell Biol. 1989; 109: 1947-1962Crossref PubMed Scopus (132) Google Scholar), we asked whether the ATPase activity of Hsp70 could play a role in DLK down-regulation. To this end we transfected COS-7 cells with the T7-tagged DLK expression construct either alone or together with a plasmid encoding a Hsp70 deletion mutant that lacks the ATPase domain (amino acids 120–428, Hsp70ΔABD) and measured the protein levels of DLK in these cells by Western blot analysis (Fig. 1C). As was the case for wild-type Hsp70, overexpression of Hsp70ΔABD also results in a dramatic decrease in ectopic DLK protein levels, indicating that the ATPase activity of Hsp70 is not essential to promote the down-regulation of DLK. Hsp70 Associates with Wild-type DLK in Intact Cells but Not in Vitro—As an approach to explore the mechanism by which Hsp70 down-regulates DLK protein levels, we first examined whether these proteins could associate physically in intact cells. For this purpose, COS-7 cells transiently transfected with T7-tagged DLK were subjected to immunoprecipitation with anti-T7 antibody, and the resultant immunocomplexes were examined by Western blot analysis using the anti-Hsp70 antibody (Fig. 2A). The immunoblot data reveals that endogenous Hsp70 associates with wild-type T7-DLK in COS-7 cells. Interestingly, this association appears to be regulated by DLK kinase activity, since the catalytically inactive T7-tagged DLK mutant did not co-immunoprecipitate endogenous Hsp70 (Fig. 2A). As a logical extension to these data, we next sought to analyze in vitro whether Hsp70 could interact directly with DLK. To do so recombinant His-tagged DLK, produced by in vitro translation, was incubated either alone or in combination with recombinant Hsp70. After incubation, proteins were subjected to immunoprecipitation analyses with the anti-Hsp70 antibody, and the resulting complexes were probed with either the anti-Hsp70 or anti-His antibody. The results shown in Fig. 2B indicates that His-DLK was not found in Hsp70 immunoprecipitates under these conditions. Taken together, these results suggest that the interaction observed between Hsp70 and wild-type DLK in COS-7 cells is weak or indirect and possibly involves the participation of at least another molecule that could bridge or stabilize this interaction. Hsp70 Reduces the Half-life of Nascent DLK—Given the observation that the steady-state levels of wild-type DLK were reduced in cells co-expressing Hsp70, we next asked whether Hsp70 could affect the turnover of DLK. To test this hypothesis we determined the half-life of DLK in the presence or absence of Hsp70 by using pulse-chase experiments. Cells transfected with T7-tagged DLK either alone or together with Hsp70 were metabolically labeled for 45 min with a mixture of [35S]methionine and [35S]cysteine and then chased with unlabeled medium for various time periods. Subsequently, T7-DLK was immunoprecipitated with the anti-T7 antibody, resolved in SDS-PAGE, and visualized by autoradiography. As shown in Fig. 3, similar quantities of newly synthesized T7-DLK were detected at chasing time 0 in COS-7 cells transiently co-transfected with or without Hsp70. However, in the presence of Hsp70 the half-life of T7-DLK was reduced by greater than 50%. Thus, our data indicate that overexpressed Hsp70 dramatically enhances the degradation of DLK in COS-7 cells. Wild-type but Not Kinase-defective DLK Is an Hsp90 Client Protein—A large number of signaling proteins in mammalian cells is known to associate with a multichaperone complex consisting of Hsp70, Hsp90, and different co-factors (39Wegele H. Mu¨ller L. Buchner J. Rev. Physiol. Biochem. Pharmacol. 2004; 151: 1-44Crossref PubMed Scopus (515) Google Scholar). This chaperone system plays a major role not only in the folding and maturation of client proteins but also in their degradation depending on the set of co-factors (39Wegele H. Mu¨ller L. Buchner J. Rev. Physiol. Biochem. Pharmacol. 2004; 151: 1-44Crossref PubMed Scopus (515) Google Scholar). Because wild-type T7-tagged DLK was found associated with Hsp70, we examined whether endogenous Hsp90 also forms a complex with this protein in transiently transfected COS-7 cells. By analogy to the data presented above, we found that endogenous Hsp90 could be detected in immunoprecipitates of T7-tagged wild-type but not kinase-defective DLK (Fig. 4A). Because
The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.
Mutations in the Drosophila kep1 gene, encoding a single maxi KH (K homology) domain-containing RNA-binding protein, result in a reduction of fertility in part due to the disruption of the apoptotic programme during oogenesis. This disruption is concomitant with the appearance of an alternatively spliced mRNA isoform encoding the inactive caspase dredd. We generated a Kep1 antibody and have found that the Kep1 protein is present in the nuclei of both the follicle and nurse cells during all stages of Drosophila oogenesis. We have shown that the Kep1 protein is phosphorylated in ovaries induced to undergo apoptosis following treatment with the topoisomerase I inhibitor camptothecin. We have also found that the Kep1 protein interacts specifically with the SR (serine/arginine-rich) protein family member ASF/SF2 (alternative splicing factor/splicing factor 2). This interaction is independent of the ability of Kep1 to bind RNA, but is dependent on the phosphorylation of the Kep1 protein, with the interaction between Kep1 and ASF/SF2 increasing in the presence of activated Src. Using a CD44v5 alternative splicing reporter construct, we observed 99% inclusion of the alternatively spliced exon 5 following kep1 transfection in a cell line that constitutively expresses activated Src. This modulation in splicing was not observed in the parental NIH 3T3 cell line in which we obtained 7.5% exon 5 inclusion following kep1 transfection. Our data suggest a mechanism of action in which the in vivo phosphorylation status of the Kep1 protein affects its affinity towards its protein binding partners and in turn may allow for the modulation of alternative splice site selection in Kep1–ASF/SF2-dependent target genes.
The Drosophila kep1 gene encodes an RNA binding protein related to the murine QUAKING apoptotic inducer. We have previously shown that kep1 can induce apoptosis when transfected into different cell lines. To better define the role of Kep1 in apoptosis, we generated kep1 null flies. These flies were viable, but females displayed reduced fertility, with approximately half of the eggs laid from kep1 − homozygotes failing to hatch. In addition, loss of kep1 suppressed GMR-rpr -mediated apoptosis in the Drosophila eye, and kep1 mutant flies had increased susceptibility to Escherichia coli infection. We found that Kep1 bound dredd RNA in vitro , and that extracts prepared from kep1 mutant ovaries had markedly reduced proteolytic cleavage activity toward the caspase-8 target substrate IETD-7-amino-4-trifluoromethyl coumarin. We observed increased levels of the β isoform of dredd mRNA in kep1 mutants as compared with wild-type. Taken together, our results suggest that Kep1 regulates apoptosis by influencing the processing of dredd RNA.
