Molecular Cloning and Characterization of Human - Europe PMC

Molecular Biology of the Cell Vol. 6, 161-170, February 1995

Molecular Cloning and Characterization of Human Kinectin Agnes Futterer, Gracia Kruppa, Bernd Kramer, Hilmar Lemke,* and Martin Kronket Institut fur Medizinische Mikrobiologie und Hygiene, Technische Universitat Munchen, Trogerstr. 9, 81675 Munchen, Germany Submitted October 17, 1994; Accepted January 3, 1995 Monitoring Editor: James A. Spudich

We have identified a human cDNA that is homologous to the chicken kinectin, a putative receptor for the organelle motor kinesin. The human cDNA clone hybridized to a single 4.6-kb mRNA species that codes for a protein of 156 kDa molecular mass. The predicted primary translation product contains an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two further C-terminal leucine zipper motifs. In addition, the aminoacid sequence revealed a large region (327-1362) of predicted a-helical coiled coils. A monoclonal antibody CT-1 raised against a GST-kinectin fusion protein produced a perinuclear, endoplasmic reticulum-like staining pattern in diverse cell types from different species, indicating evolutionary conservation. Monoclonal antibody CT-1 and anti-chicken kinectin antibodies cross-reacted both in Western blotting and immunoprecipitation with a 160-kDa protein, confirming the antigenic identity of this 160-kDa protein with chicken kinectin. Epitope tagging studies revealed that the nuclear localization sequence motif of kinectin is not functional. Furthermore, a truncated kinesin cDNA lacking the N-terminal hydrophobic domain revealed a nonspecific cytoplasmic staining pattern. Together the data suggest that kinectin is an integral membrane protein anchored in the endoplasmic reticulum via a transmembrane domain. INTRODUCTION It is well established that membraneous organelles and vesicles move along filamentous "tracks", the microtubules. The transport of organelles in the cytoplasm requires a special class of microtubule-associated proteins (MAPs) that serve to mediate their interaction with microtubules. One of these molecular motors is kinesin (for review see Schroer and Sheetz, 1991), an ATPase that moves vesicles unidirectionally along a microtubule, that is, drives transport toward the plus ends of microtubules (Schroer et al., 1988). Kinesin binds through its N-terminal head to microtubules and the C-terminal portion is believed to bind to the organelle surface (Hirokawa et al., 1989). Recently kinectin, a kinesin-binding protein of 160 kDa, has been purified from the chick embryo brain (Toyo* Present address: Institut fur Biochemie, Universitat Kiel, Olshausenstr. 40, 24098 Kiel, Germany. t Corresponding author.

© 1995 by The American Society for Cell Biology

shima et al., 1992). The properties of this protein suggested that kinectin functions as a kinesin receptor that is required for kinesin-dependent organelle movement. As part of an ongoing investigation of genes preferentially expressed in proliferating T-lymphocytes, we have identified a human cDNA with a gene product that can be uniformly detected in the endoplasmic reticulum (ER) of many cell types from different species. In this report, we show that this protein is the human homologue of chicken kinectin. MATERIALS AND METHODS Cell Lines and Culture The human lymphoma B-cell line Raji, the erythroleukemic cell line K562, and mouse, hamster, drosophila and mosquito cell lines were obtained from American Type Culture Collection (Rockville, MD). Cell lines were maintained in Click's/RPMI tissue culture medium (Biochrom, Berlin, Germany) supplemented with 5% fetal calf serum, 10 mM glutamine, and 50 ,ug/ml each of penicillin and streptomycin in a humidified incubator containing 5% Co2. 161

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cDNA Cloning The Agtll cDNA library was prepared from poly (A)' RNA extracted from the human Hodgkin-derived lymphoid cell line L540 (Diehl et al., 1981). After plaque purification, the cDNAs were subcloned into the pBluescript II SK+ vector (Stratagene, La Jolla, CA) for nucleotide sequencing by the dideoxy-chain-termination method (Sanger et al., 1977) using a Sequenase Sequencing kit (United States Biochemical, Cleveland, OH). To obtain the 5' end of Kinectin, several other libraries were screened including a HUT102 AgtlO library (Leonard et al., 1984), a K562 Agtll library (Clontech), and a YT CDM8 library (a generous gift of Dr. R. Robb, I.E. duPont de Nemours, Glenolden, PA). Oligonucleotides for sequencing were synthesized on a DNA synthesizer 381A, Applied Biosystems (Weiterstadt, Germany). cDNA sequences obtained were analyzed by the FASTA program of GenBank/EMBL.

Northern Blot Hybridization Analysis Total cellular RNA was prepared from cultured cells by the guanidinium thiocyanate method (Chirgwin et al., 1979). Poly (A') RNA was prepared by oligo-dT column chromatography (Aviv and Leder, 1972). Equal amounts of RNA (20 A per lane) were size fractionated by electrophoresis through a 0.8% agarose gel containing 2.2 M formaldehyde. RNA was transferred to nitrocellulose membranes (Schleicher & Schiill, Dassel, Germany) in 1ox SSC (1.5 M NaCl, 0.15 M sodium citrate). Membranes were hybridized with randomly primed 32P-labeled cDNA probes (Feinberg and Vogelstein, 1983).

Antibody Production and Purification Nucleotides 207-850 from the kinectin cDNA corresponding to amino acids 46-260 (p260) and nucleotides 1188-2838 corresponding to amino acids 373-923 (p923) were cloned in frame in pGEX-2T (Pharmacia, Uppsala, Sweden). Glutathione S-transferase fusion proteins were expressed as described by Smith and Johnson (1988). For purification of the fusion proteins, glutathione-sepharose was used according to the instructions of the manufacturer (Pharmacia). Monoclonal antibodies (mAb)1 to both p260- and p923-GST-fusionproteins were raised and two IgGl hybridomas were identified: mAb NT-1, directed against residues 46-260; and mAb CT-1, directed against residues 373-923. The antibodies were purified by protein A/G-agarose affinity chromatography (Dianova, Hamburg, Germany). The p923-GST fusionprotein was also used to prepare a polyclonal rabbit anti-Kinectin antibody.

Stable Expression of Kinectin cDNA in NIH3T3 Cells For stable expression of kinectin cDNA, the mouse fibroblast cell line NIH3T3 was transfected with the entire coding region of kinectin placed under the control of the SV40 enhancer/promoter present in the expression vector pSQ (kindly provided by Dr. P. Gruss, MaxPlanck Gottingen). For stable transfection, cells were cotransfected with BMGneo (Karasuyama and Melchers, 1988). After selection by G418, G418 resistant clones were isolated and kinectin-producing clones were identified by immunoprecipitation and immunoblotting analysis. For transient expression, kinectin cDNA was subcloned into the pEF-BOS expression vector (Mizushima and Nagata, 1990) kindly provided by Dr. S. Nagata.

Immunoprecipitation and Immunoblotting Cells (2 x 107) were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodium

'Abbreviations used: mAb, monoclonal antibody; NLS, nuclear location sequence; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PBL, peripheral blood leukocytes. 162

dodecyl sulfate [SDS]), supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml pepstatin, 1 mg/ml leupeptin, 1 mg/ml antipain, and 20 mg/ml trypsin/chymotrypsin inhibitor (Sigma Chemical Co., Munich, Germany). Insoluble material was removed by centrifugation for 30 min at 20,000 x g. For immunoprecipitations, cell lysates were incubated for 2 h at 4C with 20 ,ul of a protein A-Sepharose suspension (Pharmacia) coupled to either mAb CT-1, NT-1, anti-chick kinectin antibody 160.9.1, anti-kinesin antibody SUK-4 (both kindly provided by Dr. H. Yu, Durham, NC), or control rabbit anti-mouse IgG (Dakopatts, Glostrup, Denmark). The antibody-antigen complexes were washed extensively in RIPA buffer, subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to nitrocellulose membranes (Schleicher & Schiill). Kinectin was detected by incubating filters with either mAb CT-1 or anti-chick Kinectin antibody, followed by incubation with horseradish peroxidase-labeled goat anti-mouse IgG (1:10,000 dilution; BioRad, Richmond, CA). The antibody reactions were visualized either by an enhanced chemoluminescence detection system (ECL, Amersham, Buckinghamshire, UK) or by incubating filters in a chromogenic substrate.

Indirect Immunofluorescence Cells were grown on coverslips and fixed for 15 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS). For permeabilization, cells were treated for 5 min with 0.2% Triton X-100 in PBS. Kinectin proteins were stained by sequential incubation with anti-human kinectin antibodies followed by appropriate fluorescein isothiocyanate-conjugated goat anti-mouse IgG or goat anti-rabbit IgG second antibody (Medac).

RESULTS Isolation of a Full-length Kinectin cDNA By screening for cDNAs hybridizing to mRNA species

preferentially expressed by proliferating peripheral blood lymphocytes, we have obtained a clone (p16-1) that recognized a 4.6-kb mRNA species in human lymphocytes (Figure 1A). Interestingly, this clone also recognized similar-sized mRNA species in poly (A)+ RNA preparations from cell lines derived from monkey, mouse, hamster, and fish (Figure 1B). Because of this obvious conservation in evolution, this cDNA was further characterized. To obtain a full-length cDNA, additional human cDNA libraries (HUT102 in AgtlO, K562 Agtl 1 and YT in CDM8) were screened by plaque or colony hybridization, respectively, using clone p16-1 as a probe. As shown in Figures 2 and 3, partially overlapping clones revealed a 4623-bp contiguous sequence containing a single open reading frame (ORF) starting with an ATG initiation codon that is flanked by sequences consistent with the consensus sequence for initiation of translation (Kozak, 1986). The aminoacid sequence derived from the cloned cDNA predicted a protein of 156 kDa. This large ORF is followed by an untranslated sequence starting at nucleotide 4140. A potential polyadenylation signal (AATAAA) is located at position 4591, followed by a poly(A)+ tail starting at position 4614. At the nucleotide and at the protein level, two almost identical sequences have been deposited in the GenBank database (accession numbers: D13629 and L25616). There, Molecular Biology of the Cell

Molecular Cloning of Human Kinectin Figure 1. Northern blot analysis of kinectin mRNA expression. (A) Total RNA (20 ,ug) from resting (lane 1) and PMA/PHA-stimulated peripheral blood lymphocytes (lane 2) was separated on 0.8% formaldehyde-agarose gels, transferred to nitrocellulose membranes, and hybridized to 32P-labeled p16-1 cDNA. (B) Poly (A) RNA was prepared from indicated cell lines and analyzed by the 32P-labeled p16-1 cDNA probe. Ethidium bromide staining revealed that equal amounts of RNA were loaded.

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sequences represent randomly sampled cDNA clones from the human KG-1 myoblast cell line and from activated peripheral blood leucocytes (PBL). These two cDNA clones have not been further characterized. As to the identity of our cDNA sequences, we detected a homology of >70% to an embryonic chick brain protein, termed kinectin because of its kinesin-binding activity (Yu et al., 1995; GenBank accession number: U15617). At the protein level, the human kinectin has 71% identity to the chick kinectin in the first 700 N-terminal residues. From residue 700 to the C-terminus the identity ranged from 61% to 63%. To verify that the cDNA isolated encodes the human homologue of the chick kinectin, we looked for a possible antigenic relationship. mAbs were raised against the fusionprotein GST-p260 representing the N-terminal aminoacid residues 47-260 (mAb NT-1). A fusionprotein GST-p923 representing the central aminoacid residues 373-923 was used to raise both monoclonal (mAb CT-1) and polyclonal antibodies (Figure 6). Cellular extracts were immunoprecipitated with mAb CT-1 and NT-1 as well as with anti-chick kinectin antibody 160.9.1 (kindly provided by Dr. H. Yu). Immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting using either anti-chick kinectin antibody 160.9.1 or mAb CT-1. As shown in

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Figure 4, anti-chick kinectin antibodies recognized CT-1 and NT-1 immunoprecipitates of 160 kDa and 120 kDa and, vice versa, CT-1 developed similar-sized proteins previously immunoprecipitated with antichick kinectin antibody 160.9.1. Notably, an anti-kinesin antibody SUK-4 (kindly provided by Dr. H. Yu) did not recognize CT-1 or 160.9.1 immunoprecipitates. The cross-reactivity of CT-1 and 160.9.1 antibodies confirmed that our cDNA clone encodes the human homologue of chicken kinectin.

Structural Properties of Human Kinectin Hydrophobicity analysis by the method of Novotny and Auffray (1984) revealed a hydrophobic region at the very N-terminus encompassing residues 3 to 33 (Figure 5), suggesting that kinectin is a membraneanchored protein. Adjacent to this hydrophobic stretch is a bipartite nuclear targeting sequence (NLS) extending from residue 42 to residue 58 (Figure 6). Similar motifs of two basic clusters separated by 10 amino acids are found in the targeting sequences of Xenopus nucleoplasmin and in a number of nuclear proteins (Robbins et al., 1991). An unusual property of the N-terminal part of kinectin is the high proline content, that is, 19 prolines are present in the first 256 residues. These prolines are likely to produce an extremely rigid structure. A further salient feature of kinectin is the presence of sequences that fit the consensus for a dimer-forming region, termed "leucine zipper" located between aminoacid residues 934-962. The N-terminal region of kinectin (amino acids 1-200) is extremely basic (pl > 10), whereas the central and C-terminal parts appear rather acidic (pl < 5.2).

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Figure 2. Schematic representation of cDNA clones yielding the full-length kinectin cDNA. Vol. 6, February 1995

Expression of Kinectin cDNA in NIH3T3 Cells Immunoprecipitations with mAb CT-1 of extracts from actively proliferating Raji cells yielded two protein species of approximately 160 kDa and 120 kDa molecular mass, whereas mAb NT-1 recognizes only the 160 kDa protein (Figure 4). To demonstrate that both the 160-kDa and the 120-kDa species indeed de163

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rive from the same cDNA, we expressed the human kinectin cDNA in mouse NIH3T3 cells. In these cells, mAb CT-i only recognizes a single kinectin species of 150 kDa, which can thus be distinguished from the human 160-kDa kinectin (Figure 7, lane 2). These species differences allowed us to detect human kinectin expression in NIH3T3 cells directed by a human kinectin cDNA expression vector. The entire ORF of kinectin cDNA was stably expressed in NIH3T3 cells. Extracts from NIH3T3 transfectants were subjected to SDSPAGE, and immunoblotting was performed using mAb CT-i. As shown in Figure 7 (lane 3), recombinant kinectin cDNA directs expression of two protein products of 160 kDa and 120 kDa, which recapitulates endogenous kinectin expression in Raji cells. The 120kDa species may represent a degradation product of the 160-kDa form.

When a truncated kinectin cDNA lacking the N-terminal hydrophobic domain (Figure 9b) was expressed in COS cells, the structural staining pattern changed into a uniform cytoplasmic immunofluorescence. This observation supports the idea that this hydrophobic region functions as a transmembrane domain by which kinectin anchors into membranes. This staining pattern, however, suggests that kinectin is not expressed in the nucleus, and that the nuclear location sequences are not functional. To investigate the N-terminal NLS motif, the kinectin cDNA was truncated at position 1140 and tagged by a T7 major capsid protein epitope (T-Tag, Novagen, Madison, WI) (Figure 9c). As a control, the NLS motif was mutated by substitution of the lysin and arginine residues (Figure 9d). Neither of these constructs, expressed in COS cells, revealed a nuclear staining pat-

Subcellular Localization of Kinectin The presence of an N-terminal hydrophobic region (Figure 5) suggested that kinectin might be an integral membrane protein. Furthermore, like the chick kinectin, the human kinectin also proved relatively resistant to extraction by high salt or nonionic detergents (our unpublished observations), suggesting that kinectin behaves like an integral protein of the lipid bilayer, which is consistent with its putative role as organelle receptor for kinesin-directed microtubule interaction. To determine the subcellular localization of kinectin, NIH3T3 fibroblasts were stained with the polyclonal anti-kinectin antibody. As shown in Figure 8, a perinuclear, ER-like immunofluorescence pattern can be detected. Overexpression of kinectin cDNA in COS cells revealed cytoplasmic but not nuclear staining.

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Figure 4. Cross-reactivity of antibodies specific for human and chicken kinectin. Extracts from COS cells were immunoprecipitated with antibodies specific for kinesin (SUK-4, lane I), chicken kinectin (160.9.1, lane 2), or human kinectin (CT-I and NT-I, lanes 3 and 4). Immunoprecipitates were subjected to SDS-PAGE, transferred to nitrocellulose filters, and stained with either anti-chicken kinectin antibody 160.9.1 (A) or anti-human kinectin mAb CT-I (B).

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Figure 5. Hydropathic index of kinectin along its sequence according to the method of Novotny and Auffray (1984).

tern analyzed by either mAb NT-1 or monoclonal anti-T7 antibody (Novagen) (Figure 10).

DISCUSSION We herein describe a cDNA encoding a previously unknown human Ag of 156-160 kDa molecular mass.

NH2

NLS (42-58)

LZ (934-962)

j I.

I

(46-260)

mnAb

NT-1 (46-260)

COOH 1000

500 CT-1 (373-923)

By several criteria, this novel Ag appears to be kinectin, a putative receptor for the molecular motor kinesin. At the aminoacid level, this protein shares greater than 60% homolgy to embryonic chick brain kinectin, which has been identified as a major kinesin-binding protein on ER (Toyoshima et al., 1992). Further, antichicken kinectin antibody recognized the human kinectin and, vice versa, anti-human kinectin antibodies recognized the chicken kinectin, which antigenically identifies the cloned gene product as the human homologue of the chicken kinectin. Finally, human and chicken kinectin share both biochemical properties and ER-like distribution consistent with their function as an organelle receptor for kinesin. The structural features of the human 160-kDa kinectin include an N-terminal hydrophobic region. Overexpression of a kinectin deletion mutant lacking the hydrophobic domain did not reveal an ER-like distribution but instead produced a nonspecific cytoplasmic staining pattern. This observation indicates that 166

this hydrophobic region functions as a transmembrane domain and determines the subcellular distribution of kinectin. It has been previously noted that in unextracted vesicles there is a twofold molar excess of the 160-kDa kinectin over kinesin (Toyoshima et al., 1992). Correspondingly, it has been previously suggested that 160-

1356 (AA)

Figure 6. Schematic representation of kinectin indicating the relative size and position of different structural motifs. NLS, nuclear location signal; LZ, leucine zipper. The hatched area represents basic amino acid residues 1-200. The peptides used to raise mAb NT-i and CT-i are indicated.

kDa proteins at the ER form oligomers (Hurtley and Helenius, 1989). Strikingly, human kinectin contains domains for protein dimerization, which, according to NIH3T3

0. k0 OL~ kDa ra 4 205 am m"

;y

coc

I'm

116 1

2

3

Figure 7. Expression of human kinectin cDNA in NIH3T3 cells. Lysates from Raji cells or NIH3T3 cells transfected with pSQ alone or with kinectin expression vector pSQkinectin were subjected to SDS-PAGE, transferred to nitrocellulose filters, and probed with mAb CT-i. Immunoreactive proteins were detected with peroxidase-conjugated goat anti-mouse second antibody. Molecular Biology of the Cell

Molecular Cloning of Human Kinectin

Figure 8. Subcellular localization of Kinectin. Indirect immunofluorescence analysis was performed with NIH3T3 (a, b) and COS cells (c-f). COS cells were transiently transfected with either full-length (c, d) or N-terminally truncated (e, f) kinectin cDNA (see Figure 10, a and b). Polyclonal anti-kinectin antibody (b, d, f) was used as a first reagent followed by a fluorescent-labeled second antibody. This second antibody produced only weak background fluorescence (a, c, e).

Vol. 6, February 1995

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A. Futterer et al.

A

Kinectin 4300

Bp 70

1356

AA 1

Kinectin A 260

B

Bp 850

1356

AA 260

NLSwt-T7

C

Bp 70

1140T7-Tag

AA1 41

356

\

44 Gln Lys Arg Glu

CAG AAA AGA GAA

hEF1 alpha Promoter

D

NLS mut-T7 114017-Tag

Bp 70

AAI 41

356 44

Gin Asn Ser Glu

CAG AAT AGC GAA

3' UTR

SV40 on

Figure 9. Kinectin expression plasmids. Full-length (A) or 5'-truncated (B) kinectin cDNA was cloned downstream of the hEFla promoter/enhancer of pEF-BOS (13). Kinectin cDNA was deleted at position 1140 and ligated in frame with a cDNA encoding the T7 epitope (C). The NLS motif was site-specifically mutated according to the protocol of the manufacturer (D). The mutations were confirmed by nucleotide sequencing. Both constructs were cloned into pEF-BOS.

the CDPROT20 dataBank, are homologous to the leucine zipper motif of the transcription factor c-jun. The presence of two leucine zipper motifs in the human kinectin favors but certainly does not prove the idea of kinectin dimerization, which may produce a high-affinity binding site for kinesin.

168

Secondary structure analysis of kinectin by the Gascuel and Golmard Basic Statistical Method (Gascuel and Golmard, 1988) revealed that this protein is composed of a series of a-helical domains of 10-30 residues with large stretches of coil and turn conformation. According to the algorithm described by Lupas et al. (1991), residues 327 to 1362 are likely to form alphahelices and coiled-coils. At the protein level, weak homology (