Phosphorylation by cdc2-CyclinB1 Kinase Releases Cytoplasmic Dynein from Membranes*

Stephen G. Addinall, Petra S. Mayr, Sandra Doyle, John K. Sheehan, Philip G. Woodman, and Victoria J. AllanDagger

From the University of Manchester, School of Biological Sciences, Manchester, M13 9PT, United Kingdom

Received for publication, December 22, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Movement of various cargoes toward microtubule minus ends is driven by the microtubule motor cytoplasmic dynein (CD). Many cargoes are motile only during certain cell cycle phases, suggesting that CD function may be under cell cycle control. Phosphorylation of the CD light intermediate chain (DLIC) has been suggested to play a crucial role in modulating CD function during the Xenopus embryonic cell cycle, where CD-driven organelle movement is active in interphase but greatly reduced in metaphase. This down-regulation correlates with hyperphosphorylation of DLIC and release of CD from the membrane. Here we investigate the role of the key mitotic kinase, cdc2-cyclinB1, in this process. We show that DLIC within the native Xenopus CD complex is an excellent substrate for purified Xenopus cdc2-glutathione S-transferase (GST) cyclinB1 (cdc2-GSTcyclinB1) kinase. Mass spectrometry of native DLIC revealed that a conserved cdc2 site (Ser-197) previously implicated in the metaphase modulation of CD remains phosphorylated in interphase and so is unlikely to be the key regulatory site. We also demonstrate that incubating interphase membranes with cdc2-GSTcyclinB1 kinase results in substantial release of CD from the membrane. These data suggest that phosphorylation of DLIC by cdc2 kinase leads directly to the loss of membrane-associated CD and an inhibition of organelle movement.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cytoplasmic dynein (CD)1 is a multiprotein complex which binds to microtubules and moves along them using energy from ATP hydrolysis. This movement is directed toward microtubule minus ends and allows CD to move and/or position other structures (cargo) within eukaryotic cells (1, 2). CD is implicated in many important processes within eukaryotic cells during interphase, including vesicular transport from the endoplasmic reticulum (ER) to the Golgi apparatus, centripetal movement of lysosomes, nuclear migration, and maintenance of organelle structure (3). During cell division, it is likely that organelle movement is inactivated (4), while CD continues to function in the formation, alignment and maintenance of mitotic spindles, as well as the movement and positioning of chromosomes (5, 6).

The CD complex consists of four distinct classes of proteins: heavy, intermediate, light-intermediate, and light chains. The ~500-kDa cytoplasmic dynein heavy chain (DHC) is present in the complex as a dimer and is responsible for microtubule binding, ATP hydrolysis, and movement along microtubules. Two or three dynein intermediate chains (DICs) per CD complex bind to a specific region toward the N terminus of the DHC (7). The DIC also binds to the p150Glued protein, a subunit of another multi-protein complex called dynactin (reviewed in Ref. 8), and this interaction is thought to be required for many if not all of the cellular functions of CD (9). A variety of dynein light chains have been identified (10-12) and may have a role in the targeting of particular CD complexes to distinct cargo (13, 14). Four to five dynein light-intermediate chains (DLICs) are present per CD complex, binding to an N-terminal portion of the DHC near to the DIC binding site (15). DLICs show great diversity owing to the presence of two genes (DLIC1 and DLIC2), multiple spliced variants, and differential phosphorylation (15-21).

Little is known yet about the function of the DLICs. They are hyperphosphorylated in extracts made from Xenopus eggs arrested in meiotic metaphase (19). In a well characterized in vitro motility assay (22), this cell cycle-specific hyperphosphorylation of the DLIC correlated with a reduction in CD-driven membrane movement and a reduction in the amount of CD (and its accessory complex dynactin) on cargo membranes (19). On the basis of these data it has been postulated that DLICs have the following regulatory role. Mitotic hyperphosphorylation of the DLICs is proposed to cause CD to detach from cargo membranes, thus down-regulating membrane movement during mitosis. Dell et al. (23) recently demonstrated that purified cdc2 and cyclinB1 could phosphorylate recombinant chicken DLIC1 in vitro. They identified the in vitro phosphorylation site, a conserved cdc2-cyclinB1 phosphorylation motif, and showed that mutation of the phosphorylated serine residue caused the recombinant DLIC1 to become refractory to phosphorylation in meiotic cytosol (23).

Here we provide further evidence to support the hypothesis that phosphorylation of DLIC by a mitotically active kinase plays a key role in metaphase inhibition of membrane movement (19). First, we show that incubation with purified Xenopus cdc2-GSTcyclinB1 kinase removes CD from Xenopus interphase membranes in vitro. Second, we demonstrate for the first time that Xenopus DLIC within the native CD complex is specifically phosphorylated by Xenopus cdc2-GSTcyclinB1 kinase. Finally, we find that this phosphorylation occurs at a site distinct from that proposed by Dell et al. (23). Our results therefore implicate alternative conserved phosphorylation sites in the cell-cycle regulation of CD-driven membrane movement.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of Xenopus DLIC1 cDNA-- Degenerate primers DEG2I, 5'-cgggatccga(a/g)aa(a/g)ga(t/c)gc(t/c/a)gtItt(t/c)at(t/c/a)cc-3' and DEG4REVI and 5'-ggaattcIcc(a/t/g)gg(g/a/t)gaIcc(a/t/g)gt(t/c)tt(t/c)tt(a/g)ct-3' (MWG Biotech Ltd, Milton Keynes, UK) were used to amplify DLIC sequences from a Xenopus oocyte cDNA library (24). The product was cloned into pBluescriptSK+ (Amersham Pharmacia Biotech; plasmid pEB3), sequenced (Big-Dye Terminator Cycle Sequencing Ready Reaction, PE Applied Biosystems, Warrington, UK), and found to encode part of a DLIC. The pEB3 insert was used as a probe to screen the Xenopus oocyte cDNA library as per the manufacturer's instructions (Gene Images, Amersham Pharmacia Biotech). Four independent clones encoding all or part of the same novel DLIC sequence were obtained. Clone 70/1 was chosen for subsequent manipulations.

Purification of His6-XDLIC1-- Full-length N-terminal hexahistidine-tagged Xenopus DLIC1 (His6-XDLIC1) was constructed using primers BgNdLIC, 5'-gaagatctcatatggcatccgggcgtagc-3', and rseq4, 5'-taaccagcttacagaggc-3', and clone 70/1 as a template. The polymerase chain reaction product was cloned into pQE30 (Qiagen Ltd, Crawley, UK) to give pEB8. The BamHI/PstI fragment from pEB8 was replaced with the corresponding fragment from clone 70/1 to form pEB10, and the remaining polymerase chain reaction-generated DNA was sequenced. His6-XDLIC1 was purified from insoluble inclusion bodies under denaturing conditions using a His-Trap nickel column (Amersham Pharmacia Biotech).

Antibody Production and Purification-- The pEB3 insert was cloned into pGEX-4T-3 (Amersham Pharmacia Biotech) (plasmid pEB5a) to express an N-terminal GST fusion protein (GST-XDLIC1'). GST-XDLIC1' was purified as per the manufacturer's instructions (Amersham Pharmacia Biotech) and used to generate antibodies in sheep (Diagnostics Scotland, Carluke, UK). Crude serum from all bleeds (but not the pre-immune serum) recognized Xenopus DLICs. To remove contaminating anti-GST antibodies, serum was pre-absorbed over a GST column, then affinity-purified on a GST-XDLIC1' column. Eluted fractions were stored at 4 °C (with 0.02% sodium azide) and used in immunoblotting experiments at dilutions of 1/500 to 1/1000. For experiments where it was critical to remove GST antibodies completely, an alternative, small scale affinity purification method was used. The C-terminal 382 amino acids of XDLIC1 were N-terminally His6-tagged in pQE31 (Qiagen Ltd; pEB7). The fusion protein (His6-XDLIC') was enriched by isolation of insoluble inclusion bodies using B-PER (Pierce & Warriner (UK) Ltd., Chester, UK), subjected to SDS-PAGE, transferred to nitrocellulose membrane (Protran, Schleicher and Schuell), and visualized using Ponceau S (0.2% in 3% trichloroacetic acid). The His6-XDLIC' band was excised and used to affinity purify anti-XDLIC antibodies as described (25). Both types of affinity-purified antibody and the crude serum were able to immunoprecipitate Xenopus DLICs under denaturing conditions and the intact CD complex under native conditions.

Sequence Analysis-- Internet sites (NCBI, CNRS, IMGEN, EXPASY) were used for sequence analysis (26-28).

Xenopus Egg Extracts and Membrane Preparation-- Interphase and meiotic metaphase II (cytostatic factor-arrested (CSF) extracts) Xenopus egg extracts were prepared as described (29, 30). CSF extracts were converted to interphase by incubation with 0.2 mM calcium and 100 µg/ml cycloheximide for 45 min (29, 30). Membranes were floated from 200 µl of interphase extract and collected in a volume of 50 µl, as described by Lane and Allan (31) except low salt acetate buffer (20 mM potassium acetate, 3 mM magnesium acetate, 2 mM EGTA, 5 mM HEPES, pH 7.4, with KOH) replaced acetate buffer where mentioned. For Fig. 2B, membranes were floated from 40 µl of extract.

Purification of Xenopus Cytoplasmic Dynein and cdc2-GSTcyclinB1 Kinase-- CD was purified from interphase or CSF extracts as described previously (19). Bacterially expressed GSTcyclinB1 was purified and used to isolate active cdc2-GSTcyclinB1 kinase from Xenopus egg extracts as described (32). For membrane incubation experiments cdc2-GSTcyclinB1 kinase was prepared similarly except that detergent was omitted from the final two washes and elution. Histone kinase activity was assayed as described (32).

In Vitro Phosphorylation-- cdc2-GSTcyclinB1 kinase (5 µl; histone kinase activity: 50-100 pmol of phosphate incorporated into histone per µl of kinase preparation per min) was incubated with 9 µl of purified CD at room temperature for 20 min in the presence of 250 µM ATP (containing 5 µCi of [gamma -32P]ATP), 10 µg/ml protease inhibitors (PIs: stock solution 10 mg/ml leupeptin, pepstatin, chymostatin, and aprotinin in Me2SO) and 1 mM dithiothreitol in a total volume of 20 µl. Incubations were quenched with 1 ml of immunoprecipitation buffer (80 mM sodium beta -glycerophosphate, 10 mM sodium pyrophosphate, 10 mM Tris/HCl, pH 7.5, 1% Surfact-Amps X-100 (TX100; Pierce & Warriner (UK) Ltd.), 1 µg/ml PIs), incubated on ice for 10 min, then mixed with 7 µl of anti-dynein intermediate chain antibody 70.1 (Sigma) pre-bound to 40 µl of rProteinL-agarose (Actigen, Cambridge, UK). This was rotated at 4 °C for 2 h, then supernatant was removed, and beads were washed with immunoprecipitation buffer (4 × 500 µl), immunoprecipitation buffer containing 500 mM NaCl (1 × 500 µl), and 50 mM Tris, pH 7.5, containing PIs and 1 mM dithiothreitol (1 × 500 µl). SDS-PAGE sample buffer was added to the final beads, samples were subjected to SDS-PAGE, and the dry gel was exposed to x-ray film. Immunoprecipitation of CD from in vitro phosphorylation reactions was necessary because GSTcyclinB1 was autophosphorylated by the cdc2-GSTcyclinB1 kinase and runs close to DLICs on gradient gels.

Incubation of cdc2-GSTcyclinB1 with Membranes-- Interphase membranes (~15 µg of total protein) were added to 1 µl of cdc2-GSTcyclinB1 kinase (activity ~100 pmol/µl/min) in the presence of 1 mM ATP and 0.5 µM microcystin LR (Alexis Corp. Ltd, Nottingham, UK) in a total volume of 25 µl of low salt acetate buffer plus 150 mM sucrose and incubated for 30 min at room temperature. Kinase elution buffer (32) without detergent was used in place of cdc2-GSTcyclinB1 kinase as a control. Each incubation was pipetted into 25 µl of ice-cold low salt acetate buffer containing 150 mM sucrose, 10 µg/ml PIs, and 1 mM dithiothreitol, then layered onto and pelleted through a 150-µl cushion (0.5 M sucrose in low salt acetate buffer containing 0.5 µM microcystin LR, 10 µg/ml PIs, and 1 mM dithiothreitol) in a TL-100 benchtop centrifuge (TLS55 rotor, 32,000 rpm, 4 °C, 5 min; Beckman Instruments). Pelleted membranes were collected by careful removal of the cushion and addition of SDS-PAGE sample buffer, then subjected to SDS-PAGE and transferred to nitrocellulose. Immunoblotting was carried out with anti-dynein intermediate chain (monoclonal antibody 1618, Chemicon International Ltd., Harrow, UK; 1/4,000 dilution) and anti-ribophorin (CEL5C culture supernatant; a gift from Prof. Birgit Lane, University of Dundee, Dundee, UK; 1/20) followed by 35S-labeled anti-mouse IgG secondary antibody (ICN Biomedicals Inc., Basingstoke, UK), which was diluted 1/250 in Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH 7.7) containing 1% TX-100 and 1% skimmed milk powder (Marvel, Premier Brands UK Ltd, Wirral, UK). Detection was carried out using a BAS2000 imager (Fuji Photo Film Co., Tokyo, Japan), then densitometric analysis was carried out (AIDA 200, Version 2.0). Standard curves for DIC and ribophorin antibodies were obtained using a dilution series of floated interphase Xenopus membranes, which were run beside each experiment. The relative amount of DIC in each test sample was calculated by comparison with the DIC standard curve, after which this value was normalized for the amount of membrane (calculated by comparison with the ribophorin standard curve).

Analysis of DLIC Phosphorylation by Mass Spectrometry-- Tryptic digests of recombinant or endogenous DLIC were analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) using a model VG TofSpec E mass spectrometer. Recombinant His6-XDLIC (~15 µg) or purified Xenopus CD prepared from interphase or CSF extracts were subjected to SDS-PAGE and silver-stained (33), and the appropriate bands were excised from the gel. These were digested with trypsin (Promega Corp.), and the resulting peptides were processed for MALDI-MS as described (33), except that products of the enzymatic digestion were passed over Poros resin (Applied Biosystems, Cheshire, UK) before spotting onto the metal target. To obtain larger amounts of DLIC peptides for this analysis we also immunoprecipitated CD directly from crude Xenopus egg extract using anti-XDLIC1 antibody. This was carried out essentially as described (19) except that rProteinG-agarose (Zymed Laboratories Inc., San Francisco, CA) was used to carry the antibody. The peptide profiles of DLIC immunoprecipitated from extracts were identical to those from purified CD.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of Xenopus laevis DLIC1-- Multiple alignments of published DLIC sequences from rat (17), chicken (16), and human (GenBankTM accession number AAB88513) were used to design degenerate primers to the amino acid sequences EKDAVFIP and SKKTGSPG. These were used to amplify DLIC sequences from a Xenopus oocyte cDNA library (24). The resulting polymerase chain reaction product, which showed high similarity to published DLIC sequences, was used to screen the same cDNA library. Four independent clones were identified, all of which encoded part or all of the same sequence. This sequence, which was most closely related to DLIC1s from other organisms (Table I), was called Xenopus DLIC1 (XDLIC1). The XDLIC1 sequence is presented in Fig. 1 and is available under GenBankTM accession number AF317841.

                              
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Table I
XDLIC1 is most closely related to DLIC1 isoforms
Published DLIC protein sequences were compared with XDLIC1 and each other using the SIM program at EXPASY. RDLIC1, rat DLIC1 (GenBankTM accession number AAF22294); RDLIC2, rat DLIC2 (GenBankTM accession number AAA80334); HDLIC1, human DLIC1 (GenBankTM accession number AAD44481); HDLIC2, human DLIC2 (GenBankTM accession number AAB88513); GDLIC1, chick DLIC1 (GenBankTM accession number CAA55698); XDLIC1 (GenBankTM accession number AF317841). Numbers represent percent amino acid identity between two sequences.


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Fig. 1.   Sequence analysis of XDLIC1. cDNA sequence for XDLIC1. Stop codons (*), XDLIC1 open reading frame (uppercase), amino acid numbers (left), and nucleotide numbers (right) are indicated. cdc2 consensus phosphorylation sites are boxed, and the consensus P-loop sequence is underlined. The XDLIC' polymerase chain reaction product sequence runs from nucleotide 1011 to 1440.

Multiple Phosphorylation of Xenopus DLIC Proteins and Release from Membranes-- We have previously observed a single DLIC species by SDS-PAGE that migrates more slowly by SDS-PAGE in metaphase than interphase samples (19), probably owing to increased levels of phosphorylation. Here we have examined the SDS-PAGE mobility pattern of Xenopus DLICs in more detail (Fig. 2) using modified electrophoresis conditions that separate multiple DLIC bands and immunoblotting with an antibody to XDLIC1. Interphase extracts, prepared either by activating a meiotically arrested CSF extract with calcium (Fig. 2A, aCSF) or by making extracts from interphase eggs (Fig. 2B, left lane, top panel), showed three DLIC bands from ~63 to 67 kDa, with the middle band being the major species. This complex pattern is similar to that observed in species which have two DLIC isoforms, for example bovine (17), chick (16), and rat (17). This may indicate the presence of two Xenopus DLIC isoforms even though a Xenopus DLIC2 was not found in our library screen.


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Fig. 2.   Time course of DLIC molecular weight shift and loss of CD from membranes. A, a meiotic metaphase extract was incubated for 45 min with buffer (CSF) or with calcium (aCSF) to trigger entry into interphase, and then both samples were immunoblotted using affinity-purified antibody to XDLIC1. Separation of proteins in the 50-80 kDa region was optimized by running 8% SDS-PAGE gels until proteins under 50 kDa were at the bottom of the gel. Sizes of protein molecular weight markers are shown in kDa. B, interphase Xenopus egg extract was incubated with recombinant GSTcyclinB1. After the indicated times, total extracts (E) and floated membranes (M) were analyzed by immunoblot for XDLIC. Molecular mass markers are shown on the left in kDa. Asterisks mark the position of GSTcyclinB1. Histone kinase activity (HK) of total extract is indicated in pmol of phosphate incorporated into histone per µl of extract per min.

CSF extracts (arrested in meiotic metaphase II) showed a major DLIC band at 70-71 kDa and a minor band at 73 kDa (Fig. 2A, CSF). Interphase Xenopus egg extracts can be driven into metaphase by the addition of recombinant GSTcyclinB1, which leads to an activation of cdc2 kinase within 60 min, as monitored by histone kinase activity (Fig. 2B), and a shift in molecular weight of GSTcyclinB1 (see faint uppermost band in Fig. 2B, upper panel, recognized by residual anti-GST antibodies in the anti-XDLIC1 preparation), probably owing to its phosphorylation by cdc2 kinase (34). This metaphase state can be regarded as mitotic, since there is little remaining CSF activity (a characteristic of meiotic metaphase II extracts). In the presence of active cdc2-GSTcyclinB1 kinase, the DLIC migrated as a doublet of ~71 and 68 kDa (Fig. 2B, 60 min, upper panel), both forms migrating more slowly than the interphase species (0 min lane). The 68-kDa band was not observed in CSF extracts (Fig. 2A), perhaps reflecting incomplete activation of cdc2 after a 60-min incubation with GSTcyclinB1 (Fig. 2B) or, alternatively, that there are additional kinases active in CSF extracts such as MAP kinase (35), which might also phosphorylate DLIC. Since the DLIC bands observed in both types of metaphase extracts had higher apparent molecular weights than the interphase bands, all Xenopus DLICs appear to be phosphorylated in a cell cycle-dependent manner.

Because we observed a 68-kDa DLIC form after incubation of interphase extracts with GSTcyclinB1 (Fig. 2B) but not in CSF extracts (Fig. 2A), we wanted to determine whether the CD containing this phospho isoform was released from the membrane. Floated membrane samples were therefore prepared at various times after GSTcyclinB1 addition and analyzed by immunoblotting for DLICs. Thirty minutes after the addition of GSTcyclinB1, CD remained on the membrane with a completely interphase-like pattern of phosphorylation (Fig. 2B). By 60 min, histone kinase activity was high, DLIC migrated as ~71 kDa and 68 kDa bands in the total extract (Fig. 2B, upper panel), and both forms of DLIC had been lost from the membranes (Fig. 2B, lower panel). This indicated that release of CD requires cdc2 kinase activity and rules out a requirement for any other kinases that are active in meiotic but not in mitotic extracts.

Xenopus cdc2-GSTcyclinB1 Kinase Phosphorylates Xenopus DLICs in the CD Complex and Removes CD from Membranes in Vitro-- We next tested specifically whether Xenopus cdc2-GSTcyclinB1 kinase could phosphorylate Xenopus DLICs while they were part of the intact CD complex. Active cdc2-GSTcyclinB1 kinase was incubated with interphase CD (both purified from Xenopus egg extracts) in the presence of gamma -32P-labeled ATP. Intact CD complex was immunoprecipitated from this reaction mixture using a monoclonal antibody to DIC and examined by SDS-PAGE. Fig. 3A shows that a strongly radioactive protein band was detected at ~71 kDa corresponding to DLICs that had incorporated labeled phosphate. Despite possessing potential cdc2kinase phosphorylation sites (see "Discussion"), the other CD subunits were not substrates for cdc2-GSTcyclinB1 kinase in vitro. Immunoblotting for DLICs after incubation of CD with cdc2-GSTcyclinB1 kinase (Fig. 3B) revealed two new bands at around 71 and 68 kDa, very like the pattern observed 60 min after GSTcyclinB1 was added to interphase extract (Fig. 2B). None of the lowest mobility interphase DLIC remained, whereas some DLIC still co-migrated with the upper interphase DLIC form.


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Fig. 3.   Purified cdc2-GSTcyclinB1 kinase phosphorylates DLIC and removes CD from membranes in vitro. A, Xenopus CD and cdc2-GSTcyclinB1 were incubated together in an in vitro phosphorylation experiment as described under "Experimental Procedures" (lane 1). Controls were as follows: lane 2, no antibody for immunoprecipitation from the reaction mixture; lane 3, no cdc2-GSTcyclinB1; lane 4, no cdc2-GSTcyclinB1 or antibody; lane 5, no CD; lane 6, no dynein or antibody. A 5-20% gradient gel was used to ensure resolution of all CD components, but under these conditions DLIC migrates as one band. B, a similar in vitro phosphorylation experiment run on an 8% gel was immunoblotted with anti-XDLIC1 antibody. The equivalent to lanes 3 (-cdc2) and 1 (+cdc2) in panel A are shown. Sizes of protein molecular weight markers are indicated in kDa. C, immunoblotting of membranes pelleted after incubation with (+) or without (-) cdc2-GSTcyclinB1 kinase was carried out using antibodies to DIC, with ribophorin (R) as a membrane-loading control. A representative experiment is shown. The amount of CD on membranes in this experiment (as a percentage of the buffer only control) is displayed (CD) and was calculated after normalization to the amount of membrane present, as described under "Experimental Procedures."

We wondered whether this phosphorylation of DLICs in the CD complex by cdc2 kinase could be directly responsible for removal of CD from membranes as extracts enter metaphase. To test this in vitro we incubated active Xenopus cdc2-GSTcyclinB1 with membranes isolated by flotation from interphase Xenopus egg extracts. We then pelleted membranes through a sucrose cushion and immunoblotted for the DIC (to show the amount of CD) and ribophorin (an integral membrane protein to control for equal amounts of membrane) using [35S]methionine-labeled secondary antibodies. We quantitated the blot signal very carefully versus a dilution series of membrane samples loaded on the same gel to provide a standard curve for both DIC and ribophorin antibody signals (see "Experimental Procedures"). Incubation with cdc2-GSTcyclinB1 kinase caused around half of the CD (51 ± 3.2%, n = 3 ±S.E.) to be displaced from membranes as compared with the buffer only control (Fig. 3C).

The First of Four Potential cdc2 Phosphorylation Sites Conserved in DLICs Is Constitutively Phosphorylated in Interphase Xenopus Egg Extracts-- Dell et al. (23) identify a conserved cdc2 phosphorylation site (site 1; Fig. 4A) as being responsible for changes in DLIC mobility on SDS-PAGE gels after in vitro incubation of recombinant chick DLIC1 with cdc2-cyclinB1 (23). The implication of this finding is that this site is involved in cell cycle regulation of CD function. However, by comparing the XDLIC1 sequence with other published DLIC sequences and using consensi described by Pearson and Kemp (36), we have found three additional conserved potential cdc2 phosphorylation sites in DLICs (sites 2-4; Fig. 4B).


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Fig. 4.   Conserved potential cdc2 phosphorylation sites in DLICs. Accession numbers and abbreviations for sequences are as for Table I except for: DDLIC, Drosophila DLIC (GenBankTM accession number AAF48011); MDLIC1, mouse DLIC1 (partial translation from GenBankTM accession number AA146205); and MDLIC2, mouse DLIC2 (partial translation from GenBankTM accession number A253855). The following consensus sequences were used to identify potential cdc2 phosphorylation sites: (S/T)PX(K/R); (K/R)(S/T)P; and (S/T)P(K/R) (36). A, the region corresponding to amino acids 184-210 of XDLIC1 contains site 1, SPQR, which is conserved in all DLICs (SPVK in Drosophila) (23). B, the region corresponding to amino acids 379-405 of XDLIC1 contains site 2 SPR in DLIC1s (SPAR in DLIC2s, not present in Drosophila), site 3 SPR (SPLR in Drosophila) which is conserved in all DLICs, and site 4 TPxR, which is conserved in all DLIC1s.

To try to determine whether any of these sites are phosphorylated by cdc2, we isolated native CD from metaphase and interphase extracts either by classical microtubule affinity and sucrose-gradient purification or by immunoprecipitation. The DLIC proteins (and bacterially expressed His6-XDLIC1 as a control) were excised from silver-stained gels and processed for analysis by MALDI-MS. Bacterially expressed His6-XDLIC1 gave a consistent pattern of tryptic peptides, most of which were identifiable in both metaphase and interphase preparations.2 However, we found that DLICs from both interphase and metaphase CD lacked the peptide containing site 1 (2124 Da), which was obvious in the control bacterially expressed sample (Fig. 5). Instead, both traces showed a peptide 80 Da larger than the expected size (2208-2211 Da) that was absent from the control, corresponding to the addition of one phosphate to the site 1 peptide (37). We conclude that site 1 (Fig. 4A) is phosphorylated in both interphase and metaphase and therefore cannot be responsible for the regulation of CD function in this system. We therefore propose that cell cycle-dependent regulation of CD-driven membrane movement is mediated by phosphorylation of DLICs by cdc2 kinase at an alternative conserved potential phosphorylation site (Fig. 4B). The close proximity of these potential phosphorylation sites (Fig. 4B) and the lack of convenient protease sites in this portion of the XDLIC1 meant that we were unable to determine which of these alternative sites is phosphorylated using MALDI-MS.


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Fig. 5.   Analysis of trypsin-generated fragments of recombinant and native Xenopus DLICs. Peptide peaks from the 1900-2500-Da region of a MALDI-MS trace are shown from recombinant His6-XDLIC1 (A), interphase DLICs prepared by immunoprecipitation (B), and metaphase DLICs prepared by microtubule affinity (C). Peaks are labeled with their molecular mass in Da, and relative intensity of peaks is displayed at the side. The asterisk marks a peak of around 2113 that was also present in lanes containing antibody alone.


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Surprisingly little is known about how microtubule-based motors are regulated. Even in melanophores, where phosphorylation controls the direction of organelle movement, the molecular details of how this is achieved are unclear (38, 39). One example where motor phosphorylation has been directly correlated with altered motor function is the cell cycle regulation of CD-driven ER movement in Xenopus egg extracts (19). On conversion from interphase to metaphase, both ER motility and the amount of membrane-associated CD decreased ~10-fold, whereas phosphate incorporation into the DLIC increased 12 times (19). This suggests that DLIC phosphorylation by a cell cycle-regulated kinase causes CD release from membranes and, hence, an inhibition of membrane movement. The data obtained in this study support and extend this model. We have demonstrated that cdc2 kinase not only phosphorylates DLIC within the native CD complex but also triggers the release of CD from isolated interphase membranes.

Interestingly, DLIC was the only CD subunit phosphorylated by cdc2 kinase either in vitro (Fig. 3A) or in metaphase extracts (19) despite the fact that DHC and DIC sequences contain multiple potential cdc2 kinase phosphorylation sites. We can therefore conclude that there is no phosphate turnover and therefore no 32P incorporation in vitro at any of these sites in DHC or DIC in metaphase or interphase extracts. Whether these sites are unused or whether they are stably phosphorylated is not known. The latter phenomenon has been elegantly demonstrated for DIC, which becomes hyperphosphorylated upon entry into meiotic metaphase I in Xenopus oocytes and remains stably phosphorylated through multiple interphases and metaphases, at least up to stage X embryos (40). The p150Glued subunit of dynactin also behaved in this unusual manner (40), and it along with the rest of the dynactin complex is a poor substrate for cdc2 kinase in vitro.3 These data raise interesting questions about potential differential regulation of CD phosphatases during oogenesis, early embryogenesis, and the somatic cell cycle.

A key question is which if any of the four obvious potential cdc2 kinase phosphorylation sites in DLIC is used? Dell et al. (23) demonstrate that site 1 in recombinant chick light intermediate chain was efficiently phosphorylated in metaphase Xenopus egg extracts but not interphase extracts and was also phosphorylated by purified cdc2 kinase. However, when we used MALDI-MS to investigate native CD isolated from both interphase and metaphase extracts, we found that site 1 (Fig. 4) is phosphorylated regardless of cell cycle status (Fig. 5). That this site remains phosphorylated in interphase, when very little DLIC phosphate turnover takes place (19), strongly suggests that site 1 is stably phosphorylated in the native molecule and therefore cannot be responsible for the regulation of CD binding to membranes. Whether this phosphate turns over during meiotic metaphase II or whether it becomes stably phosphorylated during meiosis I, as is the case for DIC (40), remains to be established. In addition, although site 1 in recombinant protein is used by cdc2 kinase, it is also a consensus site for MAP kinase (36), and so at present, we can not conclude which kinase is responsible for its phosphorylation in the native complex in CSF extracts, which have high MAP kinase activity (35).

If site 1 can be ruled out, then what about the other three sites? cdc2 kinase sites 2-4 lie in regions that unfortunately have not generated suitable peptides for MALDI-MS with any of the proteases tested so far, so we do not yet know which site in DLIC is responsible for the observed cell cycle regulation. It is interesting to note, however, that Drosophila has only a single DLIC that appears equally related to DLIC1s and DLIC2s and that only possesses sites 1 and 3 (Fig. 4), suggesting that site 3 is the most likely candidate. One method for testing the importance of sites 2-4 is site-directed mutagenesis, but the difference between our results and those of Dell et al. (23) raises concerns over that approach. First, bacterially expressed DLIC may not be in a native conformation, either because of folding problems or because it is not assembled as part of the CD complex. Second, our preliminary results suggest that recombinant DLIC added to extracts does not become incorporated into existing Xenopus CD molecules.2 Third, bacterially expressed DLIC will be completely unphosphorylated, which may leave sites accessible that are normally stably phosphorylated. Fourth, it is well known that some phosphorylation events require other sites to be phosphorylated first, which could complicate interpretation of mutagenesis studies. It seems likely then that a combination of experimental approaches will be needed to investigate fully the regulation of CD activity by DLIC phosphorylation.

Inhibition of ER movement in metaphase egg extracts correlates with the loss of CD from the membrane (19). We show here that incubating interphase egg membranes with Xenopus cdc2-cyclinB1 kinase leads to the release of about half of the CD, suggesting that cdc2 kinase is directly involved in the cell cycle regulation of ER motility in Xenopus early embryogenesis. The simplest model is that DLIC phosphorylation by cdc2 kinase triggers the release of CD, although it is formally possible that DLIC phosphorylation prevents re-binding of any CD that detaches from membranes as part of an equilibrium. How cdc2 activity also leads to the loss of membrane-associated dynactin (19) is unclear, particularly since the dynactin complex itself is not a substrate for this kinase. However, we have not yet tested whether cdc2-dependent phosphorylation of any membrane components is also important. The fact that treatment of interphase membranes with cdc2 kinase releases ~50% of CD, whereas metaphase membranes have lost ~90% of their CD, may simply suggest that the assay with membranes and kinase alone is not fully optimized. This is quite likely, since not all of the DLIC underwent complete molecular weight shifts when purified CD was incubated with cdc2 kinase (Fig. 3B) compared with when the kinase was activated by adding GSTcyclinB1 to interphase extracts (Fig. 2B). It is also possible that other cytosolic factors are needed for full release.

The simple binding or release of CD from membranes is clearly not the only mechanism for regulating organelle movement however. For instance, a variety of organelles that are moving using plus end-directed microtubule motors during interphase also possess CD, presumably inactive, on their surface (31, 39, 41, 42). Moreover, CD-driven ER movement in interphase Xenopus egg extracts can be greatly stimulated without recruiting any extra motors (22).

One interesting question is whether the regulatory mechanism we have characterized operates during mitosis in somatic cells (rather than through the embryonic cell cycle, as assessed here and previously), as there are conflicting reports of whether CD remains associated with its membranous cargoes (43, 44). Since both DIC and p150Glued do incorporate phosphate during somatic mitosis but not during embryonic cycles (40), it seems likely that the regulatory system is more complex in the former, perhaps because there are extra functions for dynein in somatic cell division. It should be stressed that although the membrane movement functions of CD may be down-regulated in mitosis, it seems certain that there are other roles for this motor (see Ref. 9 for a review) that have to be stimulated on entry into mitosis, such as the interaction with NuMA (45) and those like the interaction between LIC1 and pericentrin, which may be active at all times (46, 47). To add to this complexity, there may be distinct CD complexes that contain specific assortments of subunits, which may be responsible for different CD functions (44, 47) and which could be differentially regulated. Other microtubule motors, such as Eg5 and MKLP1, may also be regulated by cdc2 kinase and other cell cycle-regulated kinases (48-51). Understanding how the cell integrates the function of these motors to generate different cellular processes and architecture throughout the cell cycle will require knowledge of the regulation of a whole range of motors, and Xenopus egg extracts offer an excellent model system for such investigations.

    ACKNOWLEDGEMENT

We thank Dr. David J. Thornton and David Knight for advice on mass spectrometry and Dr. Jon Lane and Pete Brown for helpful comments on the manuscript.

    FOOTNOTES

* This work was supported by Wellcome Trust Grant 048894/Z/96/A, Medical Research Council Co-operative Group Award G9722026, Medical Research Council Senior Fellowship G117/153 (to P. G. W.), and a Senior Fellowship from the Lister Institute of Preventive Medicine (to V. J. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF317841.

Dagger To whom correspondence should be addressed: University of Manchester, School of Biological Sciences, 2.205 Stopford Bldg., Oxford Rd., Manchester, M13 9PT, UK. Tel.: 44 161 275 5646; Fax: 44 161 275 5082; E-mail: viki.allan@man.ac.uk.

Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M011628200

2 S. Addinall, S. Doyle, and V. Allan, unpublished results.

3 V. Allan and P. Mayr, unpublished results.

    ABBREVIATIONS

The abbreviations used are: CD, cytoplasmic dynein; DHC, cytoplasmic dynein heavy chain; DIC, cytoplasmic dynein intermediate chain; DLIC, cytoplasmic dynein light intermediate chain; CSF, cytostatic factor; GST, glutathione S-transferase; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; PI, protease inhibitor; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Paschal, B. M., and Vallee, R. B. (1987) Nature 330, 181-183[CrossRef][Medline] [Order article via Infotrieve]
2. Vallee, R. B., and Sheetz, M. P. (1996) Science 271, 1539-1544[Abstract]
3. Lane, J., and Allan, V. (1998) Biochim. Biophys. Acta 1376, 27-55[Medline] [Order article via Infotrieve]
4. Robertson, A., and Allan, V. (1997) in Progress in Cell Cycle Research (Meijer, L. , Guidet, S. , and Philippe, M., eds), Vol. 3 , pp. 59-75, Plenum Press, New York
5. Suelmann, R., and Fischer, R. (2000) Res. Microbiol. 151, 247-254[CrossRef][Medline] [Order article via Infotrieve]
6. Heald, R. (2000) Cell 102, 399-402[Medline] [Order article via Infotrieve]
7. Habura, A., Tikhonenko, I., Chisholm, R. L., and Koonce, M. P. (1999) J. Biol. Chem. 274, 15447-15453[Abstract/Free Full Text]
8. Allan, V. (1996) Curr. Biol. 6, 630-633[Medline] [Order article via Infotrieve]
9. Karki, S., and Holzbaur, E. (1999) Curr. Opin. Cell Biol. 11, 45-53[CrossRef][Medline] [Order article via Infotrieve]
10. Harrison, A., Olds-Clarke, P., and King, S. M. (1998) J. Cell Biol. 140, 1137-1147[Abstract/Free Full Text]
11. King, S., Barbarese, E., Dillman, J., III, Patel-King, R., Carson, J., and Pfister, K. (1996) J. Biol. Chem. 271, 19358-19366[Abstract/Free Full Text]
12. Bowman, A., Patel-King, R., Benashski, S., McCaffrey, J., Goldstein, L., and King, S. (1999) J. Cell Biol. 146, 165-179[Abstract/Free Full Text]
13. Tai, A., Chuang, J.-Z., Bode, C., Wolfrum, U., and Sung, C.-H. (1999) Cell 97, 877-887[Medline] [Order article via Infotrieve]
14. Puthalakath, H., Huang, D. C., O'Reilly, L. A., King, S. M., and Strasser, A. (1999) Mol. Cell 3, 287-296[Medline] [Order article via Infotrieve]
15. Tynan, S. H., Gee, M. A., and Vallee, R. B. (2000) J. Biol. Chem. 275, 32769-32774[Abstract/Free Full Text]
16. Gill, S. R., Cleveland, D. W., and Schroer, T. A. (1994) Mol. Biol. Cell 5, 645-654[Abstract]
17. Hughes, S. M., Vaughan, K. T., Herskovits, J. S., and Vallee, R. B. (1995) J. Cell Sci. 108, 17-24[Abstract/Free Full Text]
18. Dillman, J. F., and Pfister, K. K. (1994) J. Cell Biol. 127, 1671-1681[Abstract]
19. Niclas, J., Allan, V. J., and Vale, R. D. (1996) J. Cell Biol. 133, 585-593[Abstract]
20. Maeda, S., Nam, S. Y., Fujisawa, M., Nakamuta, N., Ogawa, K., Kurohmaru, M., and Hayashi, Y. (1998) Cell Struct. Funct. 23, 169-178[Medline] [Order article via Infotrieve]
21. Maeda, S., Nam, S. Y., Fujisawa, M., Nakamuta, N., Ogawa, K., Kurohmaru, M., and Hayashi, Y. (1998) Cell Struct. Funct. 23, 9-15[Medline] [Order article via Infotrieve]
22. Allan, V. (1995) J. Cell Biol. 128, 879-891[Abstract]
23. Dell, K., Turck, C., and Vale, R. (2000) Traffic 1, 38-44[CrossRef][Medline] [Order article via Infotrieve]
24. Nicolás, F., Zhang, C., Hughes, M., Goldberg, M., Watton, S., and Clarke, P. (1997) J. Cell Sci. 110, 3019-3030[Abstract/Free Full Text]
25. Liu, B., Cyr, R. J., and Palevitz, B. A. (1996) Plant Cell 8, 119-132[Abstract/Free Full Text]
26. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve]
27. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389-3402[Abstract/Free Full Text]
28. Smith, R. F., Wiese, B. A., Wojzynski, M. K., Davison, D. B., and Worley, K. C. (1996) Genome Res. 6, 454-462[Abstract]
29. Murray, A. (1991) Methods Cell Biol. 36, 581-605[Medline] [Order article via Infotrieve]
30. Allan, V. J. (1998) Methods Enzymol. 298, 339-353[Medline] [Order article via Infotrieve]
31. Lane, J. D., and Allan, V. J. (1999) Mol. Biol. Cell 10, 1909-1922[Abstract/Free Full Text]
32. Mayr, P. S. M., Allan, V. J., and Woodman, P. G. (1999) Eur. J. Cell Biol. 78, 224-232[Medline] [Order article via Infotrieve]
33. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal. Chem. 68, 850-858[CrossRef][Medline] [Order article via Infotrieve]
34. Borgne, A., Ostvold, A., Flament, S., and Meijer, L. (1999) J. Biol. Chem. 274, 11877-11986
35. Ferrell, J., Wu, M., Gerhart, J., and Martin, G. (1991) Mol. Biol. Cell 11, 1965-1971
36. Pearson, R. B., and Kemp, B. E. (1998) in Protein Phosphorylation (Sefton, B. M. , and Hunter, T., eds) , pp. 65-83, Academic Press, Inc., San Diego, CA
37. Wilkins, M. R., Lindskog, I., Gasteiger, E., Bairoch, A., Sanchez, J. C., Hochstrasser, D. F., and Appel, R. D. (1997) Electrophoresis 18, 403-408[Medline] [Order article via Infotrieve]
38. Rogers, S., Karcher, R., Roland, J., Minin, A., Steffen, W., and Gelfand, V. (1999) J. Cell Biol. 146, 1265-1275[Abstract/Free Full Text]
39. Reese, E., and Haimo, L. (2000) J. Cell Biol. 151, 155-165[Abstract/Free Full Text]
40. Huang, C.-Y., Chang, C.-P. B., Huang, C.-L., and Ferrell, J. E. (1999) J. Biol. Chem. 274, 14262-14269[Abstract/Free Full Text]
41. Hirokawa, N., Sato-Yoshitake, R., Yoshida, T., and Kawashima, T. (1990) J. Cell Biol. 111, 1027-1037[Abstract]
42. Roghi, C., and Allan, V. (1999) J. Cell Sci. 112, 4673-4685[Abstract/Free Full Text]
43. Lin, S. X., Ferro, K. L., and Collins, C. A. (1994) J. Cell Biol. 127, 1009-1019[Abstract]
44. Tai, A. W., Chuang, J.-Z., and Sung, C.-H. (1998) J. Biol. Chem. 273, 19639-19649[Abstract/Free Full Text]
45. Merdes, A., Heald, R., Samejima, K., Earnshaw, W., and Cleveland, D. (2000) J. Cell Biol. 149, 851-861[Abstract/Free Full Text]
46. Young, A., Dictenberg, J., Purohit, A., Tuft, R., and Doxsey, S. (2000) Mol. Biol. Cell 11, 2047-2056[Abstract/Free Full Text]
47. Tynan, S. H., Purohit, A., Doxsey, S. J., and Vallee, R. B. (2000) J. Biol. Chem. 275, 32763-32768[Abstract/Free Full Text]
48. Sawin, K. E., and Mitchison, T. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4289-4293[Abstract]
49. Blangy, A., Lane, H. A., d'Herin, P., Harper, M., Kress, M., and Nigg, E. A. (1995) Cell 83, 1159-1169[Medline] [Order article via Infotrieve]
50. Giet, R., Uzbekov, R., Cubizolles, F., Le Guellec, K., and Prigent, C. (1999) J. Biol. Chem. 274, 15005-15013[Abstract/Free Full Text]
51. Lee, K. S., Yuan, Y. L., Kuriyama, R., and Erikson, R. L. (1995) Mol. Cell. Biol. 15, 7143-7151[Abstract]


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