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1 Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan
2 Department of Human Gene Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan
3 Biology Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Intracellular membrane trafficking requires the interaction of endocytic components with the cytoskeleton, microtubule and actin filaments, and the transport is powered by motor proteins, kinesins, dyneins and myosins (Apodaca 2001; Murray & Wolkoff 2003). It is generally believed that intracellular transport of cargos over long distances requires microtubules and their associated motors, whereas short-range transport is dependent on actin filaments and myosin motors (Langford 1995; DePina & Langford 1999). One example of a motor protein implicated in organelle and vesicle transport is myosin VI, an unconventional myosin that is ubiquitously expressed in higher eukaryotes, including nematodes, flies and vertebrates. This protein consists of an N-terminal myosin motor domain, a neck region containing a single IQ motif, and a C-terminal globular tail domain. In mammalian cells myosin VI exists in four splice isoforms, with or without large or small inserts in the tail region (Buss et al. 2001a; Dance et al. 2004).
Several studies have shown that, in mammals, myosin VI is involved in clathrin-mediated endocytosis, in intracellular transport of clathrin-coated and -uncoated vesicles, in the maintenance of Golgi complex morphology and in secretion (Buss et al. 1998, 2001b; Aschenbrenner et al. 2003; Warner et al. 2003). Consistent with these functions, subcellular locations of myosin VI are diverse, such as clathrin-coated and -uncoated vesicles, membrane ruffles and around the Golgi complex. Its minus-end directed motility along actin filaments, a unique characteristic that distinguishes class-VI myosin from the other classes of myosin (Wells et al. 1999), would confer on this protein the inward transport activity of endocytic cargo vesicles towards the center of cells, since it is believed that actin filaments have their minus-ends pointing inwards (Cramer et al. 1997; Verkhovsky et al. 1997).
The diverse functions and cellular localization of myosin VI are considered to be mediated by interaction with distinct cargo proteins (Oliver et al. 1999). A well characterized myosin VI-interacting protein is disabled 2 (Dab2), a putative tumor suppressor protein involved in the regulation of several signaling pathways (Morris et al. 2002). It has been suggested that Dab2 acts as an adapter protein that links cell surface receptors to the clathrin-mediated endocytic machinery and to the actin cytoskeleton through binding to myosin VI. Other myosin VI-binding partners, such as GAIP-interacting protein C-terminus and synapse-associated protein 97 also link myosin VI with endocytic vesicular compartments (Bunn et al. 1999; Wu et al. 2002).
Here, we report the identification and characterization of a new myosin VI-binding partner, LMTK2 (lemur tyrosine kinase 2)/KPI-2/cprk/BREK/AATYK2. BREK is a serine/threonine (Ser/Thr) kinase with a predicted N-terminal transmembrane sequence and a long C-terminal cytoplasmic tail. This molecule was originally identified as a binding protein of human Inhibitor-2 (Inh2), which together form a regulatory complex with a catalytic subunit of protein phosphatase-1 (PP1C), a conserved Ser/Thr protein phosphatase, and was therefore named KPI-2 (kinase/phosphatase/inhibitor-2) (Wang & Brautigan 2002). Another group identified a novel binding protein of cyclin-dependent kinase 5 (Cdk5) activator, p35, and designated it cprk (Cdk5/p35-regulated kinase), which is identical to KPI-2 (Kesavapany et al. 2003). The same protein was also identified by the other group through a database search for novel kinases expressed in brain, and was termed as BREK (brain-enriched kinase) (Kawa et al. 2004). Recently, it has been reported that male mice with a targeted disruption of the Brek gene are infertile as a consequence of azoospermia, suggesting an essential role of this protein during spermatogenesis (Kawa et al. 2006). However, physiological function(s) of this protein at the cellular level still remain to be elucidated.
Using a yeast two-hybrid screen and several binding experiments, we found that BREK interacts with the globular tail region of myosin VI. Immunocytochemical analyses showed that BREK localizes to perinuclear recycling endosomes, where transferrin receptor (TfR) or transfected Rab11 resides. siRNA-mediated knockdown experiments revealed that cells lacking BREK did not show any significant defects in internalization of transferrin (Tf) molecules and in transport to early endosomes, but the accumulation of Tf to perinuclear recycling endosomes is inhibited in these cells. These results show that BREK has a critical role in the transition of endocytosed vesicles from early endosomes to perinuclear recycling endosomes and also suggest an involvement of myosin VI in this pathway.
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Results |
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To identify novel intracellular myosin VI interactors, we performed a yeast two-hybrid screen of a human fetal brain cDNA library, using the tail domain of human myosin VI (residues 835–1285) as a bait. From a large-scale screen of 3.6 million transformants, 37 clones positive for the activation of three reporter genes were isolated, ten of which were independent clones encoding BREK. Among these BREK clones, the shortest fragment was a cDNA encoding amino acids 701–1503. From the same screening, we obtained four independent clones containing cDNAs of Dab2, which has previously been shown to interact with the globular tail of myosin VI (Inoue et al. 2002; Morris et al. 2002). One of these clones, which encoded amino acids 194–770 of Dab2, was further used as a positive control for interaction experiments with myosin VI.
BREK mRNA is present in various human and mouse tissues (Wang & Brautigan 2002; Kesavapany et al. 2003; Kawa et al. 2004). We performed an immunoblot analysis using a rabbit polyclonal antibody raised against a peptide corresponding to amino acids 707–914 of human BREK. This antibody detected a single band of > 250 kDa on immunoblots of cell lysates from CHO cells, transfected with HA-tagged full-length human BREK (Fig. 1A, left). Using this antibody, we identified endogenous expression of BREK in all human epithelial cell lines examined, including A431 (epidermoid carcinoma), Caco-2 (colon carcinoma), HeLa (cervical carcinoma), ARPE-19 (retinal pigmented epithelium) and ES-2 (ovarian carcinoma) cells (Fig. 1A, right).
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Secondly, we examined the in vitro interaction between the two proteins using a GST pull-down assay. Lysates from A431 cells were incubated with either GST or GST-fused globular tail of myosin VI (GST-M6-GTLS). The immunoblot by anti-BREK antibody shows that endogenous BREK in A431 lysates was pulled down by GST-M6-GTLS, but not by GST alone (Fig. 1C), indicating that BREK binds to the globular tail of myosin VI in vitro.
Finally, the interaction was confirmed by co-immunoprecipitation experiments. Full-length myosin VI and BREK were co-expressed in COS-7 cells and cell lysates were immunoprecipitated with anti-myosin VI antibody. Immunoblot analysis shows the interaction between full-length myosin VI and BREK in vivo (Fig. 1D). To exclude experimental artifacts due to over-expression and to address whether endogenous proteins can form a complex, soluble lysates from untransfected A431 cells were immunoprecipitated with anti-myosin VI antibody. As shown in Fig. 1E, a small but a significant amount of endogenous BREK was co-immunoprecipitated in samples incubated with anti-myosin VI but not with control mouse IgG1, indicating that at least a fraction of BREK and myosin VI can be present in a protein complex in vivo.
Intracellular distribution of over-expressed BREK
To gain insight into cellular functions of BREK, we first analyzed by confocal microscopy the intracellular localization of BREK–HA over-expressed in HeLa cells. In most cells, BREK–HA was distributed in a punctate pattern throughout the cytoplasm and was predominantly localized to the perinuclear region (Fig. 2A, left panels). Similar localization patterns were also observed in various cell lines over-expressing BREK–HA, such as A431, ARPE-19 and ES-2 cells (data not shown). Furthermore, the C-terminal HA-epitope did not seem to affect the distribution pattern, as very similar results were observed by immunostaining with anti-BREK antibody in HeLa cells over-expressing untagged BREK (data not shown) or, as reported previously, in COS cells over-expressing BREK (Kesavapany et al. 2003). We also found that BREK–HA lacking the transmembrane region (BREK
TM–HA) localized diffusely throughout the cytoplasm and did not show any membranous staining (Fig. 2D, left panel), indicating that the proper localization of BREK to the subcellular structures requires the transmembrane region.
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We also found that over-expression of BREK caused an alteration in the staining pattern of an early endosomal marker, EEA1. The early endosomes positive for EEA1 tended to co-accumulate with BREK–HA to a perinuclear region when compared with non-BREK-expressing cells, where EEA1-containing vesicles distributed more uniformly throughout the cytoplasm (Fig. 2A j–l). In contrast, under the same conditions, we observed no significant effect on the localization of GM130 or of MPR (Fig. 2A a–c, g–i), or on the organization of cytoskeletal structures, such as F-actin and microtubule (data not shown). Furthermore, the distributions of EEA1, or of any other markers mentioned above, were unaffected by over-expression of BREKTM–HA (Fig. 2D). Taken together, these results suggest that BREK has a role in membrane trafficking especially at early/recycling endosomes.
Intracellular distribution of endogenous BREK
We then examined the intracellular localization of endogenous BREK. Immunofluorescence analysis of untransfected HeLa cells using anti-BREK antibody showed that the intracellular distribution of endogenous BREK protein was comparable to that of over-expressed BREK–HA. Comparison of endogenous BREK staining among several human cell lines revealed that the perinuclear signal was most clearly observed in ES-2 cells (Fig. 3, left panels). Similar to BREK–HA over-expressed in HeLa cells, endogenous BREK staining was markedly coincident with TfR and with Tf at the perinuclear region (Fig. 3A–F). We also found that EEA1-positive early endosomes in ES-2 cells were more conspicuous in the perinuclear area, where endogenous BREK exhibited a partial colocalization with the perinuclear fraction of EEA1 (Fig. 3G–I). In contrast, co-staining with GM130 and BREK yielded clearly distinguishable patterns, indicating that endogenous BREK is not located at the Golgi apparatus (Fig. 3J–L). Colocalization of MPR and BREK was observed to be very limited (Fig. 3M–O). These immunocytochemical studies revealed that BREK mainly localizes in the perinuclear early/recycling endosomal components.
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To elucidate the subcellular location where BREK interacts with myosin VI, we analyzed the intracellular localization of BREK and GFP-tagged myosin VI in HeLa cells. Previous studies have shown that GFP-myosin VI is localized around the Golgi complex in the perinuclear region and also in cellular membrane vesicles, including clathrin-coated or -uncoated vesicles (Buss et al. 2001a; Aschenbrenner et al. 2003). When both BREK–HA and GFP-myosin VI constructs were co-expressed in HeLa cells, GFP-myosin VI partially colocalized with BREK–HA in the perinuclear region as well as in vesicles in the cytosol (Fig. 4A–C). In some cells, this perinuclear staining with GFP-myosin VI also overlapped with TfR (Fig. 4A–E). GFP-myosin VI and BREK–HA also colocalized with the Golgi marker GM130 in some HeLa cells. Although we could not rule out the possibility that the colocalization at the Golgi complex is due to over-expression of BREK, these results suggest that BREK executes its cellular function, when together with myosin VI, at perinuclear organelles including recycling endosomes and the Golgi complex, as well as at cytoplasmic vesicles.
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The binding property of BREK with myosin VI and the subcellular localization of BREK to the endosomal structures led us to hypothesize that this transmembrane kinase is involved in the endocytic machinery. To test this hypothesis, we performed siRNA-mediated silencing of endogenous BREK and analyzed loss-of-function phenotypes of Tf uptake and recycling. To increase confidence in data from siRNA experiments, two independent siRNA duplexes (BREK siRNA#1 and #2, see Experimental procedures) specific to the nucleotide sequences of human BREK were tested in HeLa cells. In both cases, the best results were achieved 48 h after transfection when > 90% of endogenous BREK protein was depleted, compared to the cells transfected with non-silencing control siRNA (Fig. 5A). Expression of an unrelated 
-tubulin gene product was not affected in the BREK siRNA-treated cells, confirming the specificity of the silencing. Consistent with the Western blot data, immunofluorescence analysis of BREK knocked down cells showed that an almost complete depletion of BREK staining subsequent to the siRNA treatments (Fig. 7A).
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To dissect which stage of endocytic events was impaired in BREK siRNA-treated cells, we performed detailed analysis of pulse-chase Tf uptake experiments. In these studies, siRNA-transfected HeLa cells were serum starved and surface TfRs were labeled with Alexa 546-Tf at 4 °C. In control cells, after 2-min chase at 37 °C, the majority of internalized Tf was evident as dispersed punctate patterns throughout the cell periphery (Fig. 6A b). These spots displayed a high degree of colocalization with EEA1, showing that early endosomes were labeled with Tf at this time-point (Fig. 6A a–c). Under the same conditions, the distribution of Tf in BREK depleted cells was indistinguishable from that in control cells after 2-min chase (Fig. 6A d–f). This result suggested that the early endocytic events of clathrin-mediated endocytosis, including the formation of clathrin-coated vesicles and transport to early endosomes, were not perturbed by BREK depletion.
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To further confirm the effect of BREK knockdown, we generated an siRNA-resistant BREK by introducing six silent mutations into the BREK siRNA#1 binding site of BREK cDNA. A considerable amount of siRNA-resistant BREK–HA (srBREK–HA) was successfully expressed in the BREK siRNA#1-treated HeLa cells (Fig. 8A). Importantly, re-expression of full-length BREK could rescue the knockdown phenotype, as 76.2 ± 3.0% of the srBREK–HA transfected cells exhibited the perinuclear accumulation of Tf (Fig. 8B, C). In contrast, EGFP or BREK lacking the transmembrane region failed to rescue the phenotype, as only 42.6 ± 6.7% or 34.3 ± 4.1% of the cells transfected with EGFP or with siRNA-resistant BREK–HA (srBREKTM–HA), respectively, showed the perinuclear accumulation of Tf.
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Discussion |
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BREK is a member of the AATYK kinase subfamily which is composed of LMTK1/AATYK1, LMTK2/KPI-2/cprk/BREK/AATYK2 and LMTK3/AATYK3. Unique features of the AATYK family members are the presence of N-terminal membrane-targeting sequences and unconventional catalytic sequences in the kinase domain, which exhibit higher homology to tyrosine kinases than to those of Ser/Thr kinases, regardless of Ser/Thr kinase activity (Wang & Brautigan 2002; Kawa et al. 2004; Tomomura et al. 2007). Despite the biological significance of BREK in spermatogenesis in male mice (Kawa et al. 2006), definitive functions of the members of the AATYK family at the cellular level remain largely unresolved.
Herein, we describe the characterization of BREK with regard to its cell biological properties. We provide several lines of evidence suggesting that BREK is involved in the regulation of the membrane cargo traffic from early endosomes to recycling endosomes. Immunocytochemical analyses revealed that BREK localizes to cytoplasmic vesicles and to perinuclear membranous structures through its N-terminal transmembrane region. These perinuclear structures most likely belong to recycling endosomes, as shown by colocalization with TfR or GFP-Rab11. Furthermore, the cells in which BREK was depleted by siRNA were still able to internalize Tf molecules and transport them to early endosomes, but failed to continue this transport to perinuclear recycling endosomes. This is probably due to impaired motility of membrane traffic from early endosomes to recycling endosomes, or to dysfunction of early endosomes and/or recycling endosomes in the Tf endocytic recycling pathway. In addition, we found that EEA1-positive structures co-accumulate with over-expressed BREK–HA, suggesting that BREK plays a role in maintaining early endosomes.
Our results also suggest that, of the two distinct recycling pathways, BREK is specifically involved in the slow pathway, via the perinuclear recycling endosomes. Although BREK knockdown caused a dramatic reduction of Tf accumulation in the perinuclear recycling endosomes, total Tf recycling rates were not much perturbed in the BREK-RNAi cells. Judging from these observations, the rapid recycling pathway, independent of the perinuclear recycling endosomes, seems not to be significantly affected by BREK knockdown. A similar phenotype has been reported in cells with depletion of EHD3, a member of the EHD (Eps15 homology domain) family proteins implicated in regulating endocytic recycling (Naslavsky & Caplan 2005). Our results suggest that BREK functions in the endosomal recycling route, irrespective of the rapid recycling.
Considering that BREK is a large transmembrane protein with an active Ser/Thr kinase domain, BREK could exert its function in the membrane recycling pathway by anchoring and/or actively regulating the components on recycling endosomes by phosphorylation. We identified BREK as a myosin VI-binding protein that also plays a key role in the endocytic membrane traffic pathway, and confirmed the specificity of BREK–myosin VI interaction by multiple binding experiments. Although detailed observation of the cellular location of endogenous BREK–myosin VI protein complex needs to be established, partial colocalization of BREK with over-expressed myosin VI in cytoplasmic vesicles and TfR-positive perinuclear structures suggests the function of this protein complex in membrane vesicle recycling. In fact, the idea of myosin VI-function at recycling endosomes is supported by recent studies reporting that endogenous myosin VI and optineurin are present in Rab8- and TfR-positive recycling endosomes, and that myosin VI is involved in AP-1B-dependent basolateral membrane sorting in MDCK cells (Sahlender et al. 2005; Au et al. 2007). BREK might anchor this actin-based motor to recycling endosomes and/or regulate this protein by direct phosphorylation, however, we did not detect BREK-induced phosphorylation of full-length myosin VI in vitro under our experimental conditions (Supplementary Fig. S2).
The notion of co-operation between BREK and myosin VI is further supported by recent findings in endocytosis of cystic fibrosis transmembrane regulator (CFTR), a cAMP-activated Cl- channel and also a regulator of several transporters and channels. CFTR is endocytosed from the cell surface by the clathrin-dependent pathway and then recycled back to plasma membrane through recycling endosomes in an identical manner to TfR (Picciano et al. 2003). CFTR forms a protein complex with myosin VI, and efficient CFTR endocytosis requires functional myosin VI (Swiatecka-Urban et al. 2004). Meanwhile, CFTR has been identified as a candidate kinase substrate for BREK (Wang & Brautigan 2006). Based on these studies, together with our findings suggesting a direct interaction between BREK and myosin VI, we speculate that BREK-dependent phosphorylation of CFTR and BREK/myosin VI interaction contribute to the cellular machinery required for the CFTR endocytic/recycling pathway at early/recycling endosomes.
In addition to myosin VI, other proteins that have been shown to interact with BREK are possibly involved in the membrane recycling pathway. These BREK-interacting proteins include p35–Cdk5 kinase complex and Inh2-PP1C phosphatase complex. Previous studies have revealed that they actually participate in endocytic pathways, although their detailed roles remain to be elucidated. In neurons, Cdk5 is involved in membrane fusion, secretion, and endocytosis by phosphorylation of dephosphins that include amphiphysin, dynamin 1 and Munc-18 (Shuang et al. 1998; Tomizawa et al. 2003). PP1 activity is critical for endocytic vesicular trafficking (Peters et al. 1999). Therefore, in future studies, protein phosphorylation and interaction networks between BREK and its binding proteins need to be clarified to elucidate the molecular details of endocytic trafficking, especially in the membrane recycling pathway.
BREK is essential for spermatogenesis (Kawa et al. 2006), during which many events of intracellular membrane traffic take place. Acrosome biogenesis, an important early process of spermiogenesis, relies on intense intracellular membrane trafficking/fusion (Ramalho-Santos et al. 2001). Given that Brek knockout mice had deformed acrosomes (Kawa et al. 2006), further studies on these mice could also contribute to the understanding of the molecular mechanisms by which BREK is involved in these processes.
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Experimental procedures |
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The mammalian BREK expression vectors were constructed as follows. An N-terminal portion of human BREK cDNA was obtained from an XhoI-EcoT14I fragment of KIAA1079 (Kazusa DNA Research Institute, Kisarazu, Japan). A C-terminal portion of BREK cDNA was generated by polymerase chain reaction (PCR) using a positive clone isolated from a yeast two-hybrid screen that contains the C-terminal part of BREK with a forward primer; 5'-CGT TCA CAG CTG GCT CCC AGG-3' and a reverse primer; 5'-TGC GGC CGC TAG TCC TTT TCT CCG TCT TCG CTG-3' and was digested by EcoT14I-NotI. These N-terminal and C-terminal fragments were cloned into an XhoI-NotI site of vector pCI-neo (Promega, Madison, WI). To construct a vector of BREK–HA (pCI-BREK–HA), an HA-tag was inserted into the C-terminal end of BREK by PCR. BREKTM–HA (amino acids 71–1503) was constructed by PCR with a forward primer containing a Kozak sequence and a start codon; 5'-AGC TAG CCG CCA CCA TGG ACC CAG AAA TAG ACT TTA AGG AAT TTG-3' and a reverse primer; 5'-CTT GGT TGA GGA CAT CTA AGT TGG-3'. An NheI-BstEII fragment of this PCR product was cloned into pCI-BREK–HA, digested with NheI-BstEII. siRNA-resistant BREK constructs were generated by QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using the primer 5'-CAG TCC CCA GCT GAA GTG TTT ACG CTG TCA GTA CCA AAT ATT TC-3'.
The full-length human myosin VI cDNA was obtained from clone KIAA0389 (Kazusa DNA Research Institute) by PCR and inserted into vector pCI-neo or pEGFP-C3 (BD Biosciences Clontech, Palo Alto, CA). Note that clone KIAA0389 contains both the large insert of exon bc (designated in Dance et al. (2004)) and the small splice insert.
Human Rab11a cDNA was obtained by PCR using a human fetal brain cDNA library (Invitrogen, Carlsbad, CA) with a forward primer; 5'-GGC TCG AGG GCA CCC GCG ACG ACG AGT-3' and a reverse primer; 5'-CCG CGG CCG CTT AGA TGT TCT GAC AGC ACT G-3' and was inserted into vector pEGFP-C3.
Antibodies
To generate a polyclonal antibody to BREK, a cDNA fragment encompassing residues 707–914 of human BREK was cloned into vector pGEX-4T-3 (Amersham Biosciences, Piscataway, NJ). Fusion protein GST-BREK707–914 was then expressed and purified from Escherichia coli strain BL21. Rabbit polyclonal antibody was raised by immunization with the fusion protein and affinity purified, as described previously (Buss et al. 1998). Other antibodies used were from the following sources: mouse monoclonal anti-myosin VI (MUD-19) from Sigma-Aldrich (St. Louis, MO), mouse monoclonal anti-GM130 and anti-EEA1 from BD Transduction Laboratories (San Diego, CA), mouse monoclonal anti-TfR from Zymed Laboratories (South San Francisco, CA), rat monoclonal anti-HA and fluorescein-conjugated anti-HA (3F10) from Roche (Mannheim, Germany), mouse monoclonal anti-MPR from Calbiochem (La Jolla, CA), Cy3-conjugated goat anti-mouse IgG and Cy3-conjugated goat anti-rat IgG from Jackson Immunoresearch (West Grove, PA), Alexa 488-conjugated goat anti-rabbit IgG and Alexa 488-conjugated goat anti-mouse IgG from Molecular Probes (Eugene, OR), mouse monoclonal IgG1 from Ancell (Bayport, MN).
Cell culture and transfection
HeLa, CHO-K1, COS-7 and A431 cells (RIKEN Cell Bank, Ibaraki, Japan) were cultured in DMEM, supplemented with 10% fetal bovine serum (FBS), ARPE-19 cells (American Type Culture Collection (ATCC), Manassas, VA) were maintained in DMEM-F12 with 10% FBS and ES-2 cells (ATCC) were grown in McCoy's 5A medium with 10% FBS at 37 °C in 5% CO2. For transfection experiments, cells were transfected with FuGENE6 (Roche) according to the manufacturer's instructions.
Yeast two-hybrid screen and mammalian two-hybrid assay
For the yeast two-hybrid screen, the tail region of human myosin VI (835–1285 amino acid) was inserted into vector pDBLeu (ProQuest Two-Hybrid System; Invitrogen) and used as a bait to screen the human fetal brain cDNA library according to the manufacturer's protocol. Positive clones were isolated from colonies on selection plates lacking leucine, tryptophan and histidine and containing 50 mM 3-amino-1,2,4-triazole, and were tested for the induction of HIS3, URA3 and lacZ reporter genes. The plasmids containing candidate cDNAs of interacting proteins were isolated and sequenced.
For the mammalian two-hybrid assay, the cDNAs encoding myosin VI whole tail (M6-WT; 840–1285 amino acid), coiled-coil (M6-CC; 840–1031 amino acid), globular tail with the large and small inserts (M6-GTLS; 1031–1285 amino acid), globular tail lacking the small insert (M6-GTL) or the large insert (M6-GTS) were cloned into vector pACT (CheckMate Mammalian Two-Hybrid System; Promega). The cDNA corresponding to the BREK gene (341–1503 amino acid) from one of the positive clones of the yeast two-hybrid screen was cloned into vector pBIND (Promega). Dab2 fragment (194–770 amino acid) from another positive clone of yeast two-hybrid screen was also cloned into vector pBIND. CHO cells were transfected with these vectors together with vector pG5luc (Promega) for internal control and the activities of Renilla and firefly luciferase were quantitated using the Dual-Luciferase Reporter Assay System (Promega).
Co-immunoprecipitation and GST pull-down
Cells were lysed with lysis buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40 and protease inhibitor cocktail (Complete; Roche) and centrifuged for 30 min at 20 000 g to remove insoluble materials. For co-immunoprecipitation, the soluble extracts precleared with Protein G Sepharose beads (Amersham Biosciences) were incubated with antibodies for at least 3 h. After additional incubation with Protein G Sepharose beads for 2 h, the immunoprecipitates were washed 5 times with lysis buffer.
For the GST pull-down assay, GST or GST-tagged human myosin VI globular tail (M6-GTLS, see above) were expressed in BL21 and purified with glutathione-Sepharose 4B (Amersham Biosciences). GST or GST-M6-GTLS-coated beads were incubated with soluble A431 cell lysates for 2 h and washed 5 times with lysis buffer.
Proteins precipitated with Protein G Sepharose beads or with glutathione-Sepharose 4B beads were eluted by the addition of equal volumes of 2x SDS sample buffer and were boiled for 5 min. The samples were resolved by SDS-PAGE, transferred to PVDF membrane and immunoblotted with appropriate antibodies.
Knockdown of BREK by siRNA
Cells were transfected with non-targeting control siRNA, BREK siRNA#1 (5'-GCA GAG GUC UUC ACA CUU UTT-3') or BREK siRNA#2 (5'-UAA AUG AUC UUC AGA CAG ATT-3') duplexes (Dharmacon, Lafayette, CO) at a final concentration of 100 nM using Oligofectamine reagent (Invitrogen) according to the manufacturer's instructions. The knockdown efficiency was assessed by Western blotting 2 days after transfection.
Immunocytochemistry
Cells grown on coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min followed by permeabilization with 0.1% Triton-X in PBS for 5 to 20 min and by blocking with blocking buffer containing 3% bovine serum albumin (BSA) and 1% normal goat serum (NGS) in PBS for 30 min. Then cells were incubated with primary antibodies diluted in PBS with 0.1% BSA and 1% NGS at 37 °C for 1 h, washed with PBS, and incubated with the appropriate labeled secondary antibodies diluted in PBS with 0.1% BSA and 1% NGS at 37 °C for 1 h. Washed coverslips were mounted in 80% glycerol in PBS containing 0.1 M n-propyl gallate and sealed with nail polish. Images were acquired using a Bio-Rad Radiance 2000 confocal laser scanning microscope and processed with Adobe Photoshop software.
FACS-based quantification of Tf uptake and recycling
Two days after transfection of siRNA, cells were trypsinized and resuspended in PBS with EDTA and used for FACS-based Tf uptake and recycling analyses. For Tf uptake assays, cells were incubated with serum-free media for 1 h at 37 °C and then incubated with ice cold media containing 20 µg/mL FITC-conjugated human Tf (Jackson ImmunoResearch) for 30 min at 4 °C. Samples were warmed to 37 °C for indicated time intervals in the same media to allow internalization of Tf. For Tf recycling assays, cells were incubated in serum-free media containing 20 µg/mL FITC-Tf for 1 h at 37 °C. Cells were then incubated in media with 100 µg/mL unlabeled human holo-Tf (Sigma) at 37 °C for various times. The uptake and recycling experiments were terminated by pelleting and fixing the cells in 1% paraformaldehyde. The amount of cell-associated FITC-Tf was determined by FACS analysis using a FACSCalibur (Becton Dickinson) equipped with 488-nm laser, counting for at least 1000 cells per time-point. The error bars indicate the SEM from three separate experiments.
Quantification of Tf uptake
Cells grown on coverslips were starved in serum-free media for 2 h at 37 °C. They were then incubated in serum-free media containing 20 µg/mL Alexa 546-conjugated human Tf (Molecular Probes) at 37 °C for 2 min or for 30 min, fixed and processed for immunofluorescence, as described above.
To quantify the effect of siRNA on the perinuclear accumulation of endocytosed Tf, the percentage of cells with perinuclear accumulation of Tf was calculated. The number of cells that showed a perinuclear accumulation of Alexa 546-trasnferrin was divided by the total number of cells counted. The error bars indicate the standard deviation of three independent experiments (n > 100 for each experiment).
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Acknowledgements |
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Footnotes |
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* Correspondence: Email: sutoh{at}bio.c.u-tokyo.ac.jp
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References |
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Accepted: 11 February 2008
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