(Received for publication, November 27, 1996, and in revised form, December 9, 1996)
From the We have isolated a 27-kDa protein that binds to
cytoplasmic dynein. Microsequencing of a 17-amino acid peptide of this
polypeptide yielded a sequence which completely matched the predicted
sequence of the Cytoplasmic dynein is a mechanochemical ATPase that powers
retrograde organelle transport in a wide variety of cell types and has
been implicated in many different cellular functions, including the
endocytic pathway, Golgi positioning, chromosome movement, spindle
formation, nuclear positioning, and retrograde axonal transport
(recently reviewed in Ref. 1). Both the intracellular targeting and the
motor activity of cytoplasmic dynein are likely to be tightly regulated
within the cell (reviewed in Ref. 2); however, little is as yet known
about the mechanism of this regulation.
Cytoplasmic dynein is a macromolecular complex of two heavy chains
(~540 kDa), approximately three intermediate chains (~74 kDa), and
four electrophoretically distinct light chains (~53-59 kDa). The
cytoplasmic dynein intermediate chains
(DICs)1 have been suggested to be
functionally analogous to the axonemal dynein intermediate chains in
targeting the dynein motor to its cargo (3). The dynein intermediate
chains have also been shown to link the motor enzyme to dynactin by
direct interaction with the p150Glued component of dynactin (4,
5). Dynactin is a complex oligomer of ~20 S that has been reported to
increase the frequency of vesicle movement mediated by dynein in
vitro (6), and which may provide an essential link between the
motor and the organelle (7). Regulation of the dynein-dynactin
interaction may be a key step in the mechanism of cytoplasmic
dynein-mediated organelle transport.
Casein kinase II (CKII) is a ubiquitous eukaryotic serine/threonine
kinase which is found in higher eukaryotes as either an In this study we isolated CKII as a cytoplasmic dynein-binding protein
using affinity chromatography (5). We demonstrate that CKII binds to
the intermediate chain of dynein, but is not a component of the
dynactin complex, and that CKII is capable of phosphorylating the
dynein intermediate chain in vitro. This phosphorylation of
DIC by CKII may modulate dynein function at the level of dynein complex
assembly, motor activity, or specificity of interaction with its cargo,
and potentially may play a role in modulating the interphase and
mitotic functions of cytoplasmic dynein.
Rat
brain cytosol was prepared as described previously (5) and either
loaded onto a dynein affinity column or used for cytoplasmic dynein
preparation by microtubule affinity (34). In some experiments purified
cytoplasmic dynein was treated with lambda protein phosphatase (New
England Biolabs, Beverly, MA); the phosphatase was removed by linear
sucrose density gradient centrifugation. Recombinant cytoplasmic dynein
intermediate chain was expressed in Escherichia coli and
purified as described previously (5).
Dynein intermediate chain
cross-linked to CH-Sepharose 4B beads at a concentration of ~2 mg
ligand/ml of drained beads was used for all affinity chromatography
experiments. For isolation of novel dynein-binding proteins, 10 ml of
brain cytosol was loaded onto a 2-ml dynein affinity column and the
column was washed with 50 volumes of PHEM (50 mM Na-HEPES,
50 mM Na-PIPES, 1 mM EDTA, 1 mM
MgCl2, pH 6.9) buffer. This extensive wash was followed by a 2-bed volume wash with 25 mM NaCl in PHEM. The column was
then eluted with PHEM containing 1 M NaCl and dialyzed
against PHEM containing 0.2 mM dithiothreitol, then
resolved on a 5-20% sucrose gradient. The resulting fractions were
analyzed by SDS-PAGE, and those fractions near the 20 S peak were
pooled and methanol-precipitated. For other experiments, the column
size was reduced to 0.5-ml bed volumes and processed as described above
and eluted with 1 M NaCl in PHEM. The eluate fractions were
methanol-precipitated. The precipitates were resolved by 10% SDS-PAGE,
transferred onto Immobilon-P (Millipore, Bedford, MA), and probed with
anti-human CKII Immunoprecipitation from rat brain
cytosol was carried out as described previously (17) with an
affinity-purified rabbit polyclonal anti-p150Glued
antibody. The immunoprecipitate was then resolved by SDS-PAGE, transferred to Immobilon-P, and probed with anti-p150Glued,
anti-p50, anti-centractin, and anti-CKII The methanol precipitate of the eluate
from the dynein affinity column was resolved by 10% SDS-PAGE,
transferred to Immobilon-P, and briefly stained with Ponceau S. A
polypeptide band corresponding to approximately 27 kDa was excised and
sent to Dr. J. Leszyk of the Worcester Foundation for Biomedical
Research for in situ trypsin digestion and amino-terminal
amino acid microsequencing of the resulting peptides on a 477A
Sequenator (Applied Biosystems, Foster City, CA).
Purified recombinant dynein
intermediate chain was equilibrated in PHEM buffer using a NAP-10 gel
filtration column (Pharmacia, Uppsala, Sweden). In six identical tubes
10 µg of DIC, 250 µM Mg-ATP, 10 µCi of
[ We have previously
demonstrated that a dynein intermediate chain affinity column retains
dynactin complex from brain cytosol (5). We extended this technique to
identify other potential dynein-binding proteins. Rat brain cytosol was
loaded onto an intermediate chain affinity column and after extensive
washing, the column was eluted with 1 M NaCl. Initially, we
resolved the eluate on a sucrose gradient and the fractions
corresponding to ~20 S were pooled and methanol precipitated.
Analysis of the precipitate by SDS-PAGE revealed an uncharacterized
polypeptide of 27 kDa (Fig. 1A).
Amino-terminal amino acid sequencing of a 17-residue-long tryptic
fragment of the 27-kDa band yielded the sequence IHPMAYQLQLQAASNFK. This sequence was then used in a BLAST search (18) using the NCBI data
bases. The peptide sequence completely matched the predicted protein
sequence of the well characterized
Since CKII exists as a tetramer of 2
It has been reported that
there is a 27-kDa dynactin polypeptide (20). To test the possibility
that the 27-kDa CKII
Primary amino acid sequence
analysis of the rat dynein intermediate chain revealed 11 potential
CKII phosphorylation sites. To investigate whether CKII could
phosphorylate dynein in vitro, we incubated bacterially
expressed and purified rat DIC with CKII and
[
We also tested whether dynein in its native state could be
phosphorylated in vitro by CKII. Purified cytoplasmic dynein
was incubated with recombinant CKII at 30 °C, and the reactions were analyzed by SDS-PAGE followed by autoradiography (Fig. 5B).
The intermediate chain of cytoplasmic dynein was phosphorylated, as well as the dynein heavy chain (lanes 4-6). However, the
observed extent of phosphorylation was relatively low. We hypothesized that this low level of incorporation might be because the dynein was
phosphorylated as isolated (21, 22). Therefore we pretreated the
purified dynein with lambda protein phosphatase, removed the phosphatase by sucrose gradient centrifugation, and then compared the
phosphorylation of the phosphatase-treated dynein with that of
untreated dynein (lanes 1-3). Phosphorylation of the
intermediate chain was increased by ~2-fold (as judged by
densitometric analysis) following phosphatase treatment (lane 3 versus lane 6), to a level of 0.5 mol of phosphate incorporated
per mol of the intermediate chain. Taken together, these results
suggest that CKII is capable of phosphorylating the intermediate chain
in native dynein and that the intermediate chain in the dynein complex
as isolated may already be phosphorylated at CKII-specific sites.
The intermediate chains of cytoplasmic dynein have been suggested
to play a role in the targeting of the motor (3). Recent biochemical
evidence demonstrating direct binding between cytoplasmic dynein
intermediate chains and the p150Glued component of dynactin (4,
5) also point to a potential targeting role for the intermediate
chains. Using an affinity column assay we have described previously
(5), we have now determined that the The size of the CKII The observation that CKII binds to dynein is of particular interest
since CKII has been reported to interact with a number of proteins,
some of which are thought to be physiological substrates of CKII
(23-31). For example, nucleoplasmin, a nuclear protein, associates
with and is phosphorylated by CKII (24). Similarly, FK506-binding
protein (a 25-kDa nuclear protein) also associates with CKII (31).
Finally, CKII copurifies with proteosomes and phosphorylates the 30-kDa
proteosome subunit (23). Since the CKII heterotetramer bound to dynein,
we tested whether CKII could phosphorylate dynein in vitro.
The results presented here demonstrate that bacterially expressed
dynein intermediate chain is phosphorylated by recombinant CKII. We
also show that intermediate chains in the purified dynein complex can
also be phosphorylated by CKII. The observation that pretreatment of
cytoplasmic dynein with a phosphatase increases the subsequent
phosphorylation by CKII (Fig. 4) lends support to the hypothesis that
intermediate chains in the dynein complex are phosphorylated within the
cell, potentially on CKII-specific sites.
Despite cytoplasmic dynein's potential roles in a wide range of
cellular events, very little is known about how a single major form of
cytoplasmic dynein can participate in a regulated manner in all of
these processes. One key regulatory mechanism for cytoplasmic dynein
may be its phosphorylation (see Ref. 2 for a recent review). CKII is an
ubiquitous serine/threonine kinase with an established role in cell
proliferation. While the CKII regulatory pathway is not well
understood, it is interesting to note that CKII has been shown to
localize to the spindle apparatus and centrosome; cytoplasmic dynein
has also been shown to localize to these cellular structures (1).
Dynein phosphorylation may affect the affinity of the enzyme for its
cellular cargo. Several studies have noted a correlation between dynein
phosphorylation and its distribution on membranous organelles (22, 32,
33). Phosphorylation may also affect the mechanochemical cycle of
cytoplasmic dynein. It has been reported that dynein undergoing
anterograde transport (therefore in the inactive state) is
differentially phosphorylated than the general dynein pool (21),
suggesting that the phosphorylation of dynein may regulate its motor
activity. It is possible that other polypeptides either in dynein or in
the closely associated dynactin complex are physiological substrates of
CKII. As described above, we have noted the phosphorylation of dynein
heavy chain in vitro by CKII; our preliminary observations
also suggest that some of the dynactin polypeptides are targets for
phosphorylation by this kinase.
In summary, CKII alone or in conjunction with other kinases may modify
DIC in such a way as to modulate dynein function at the level of
complex assembly, dynein activity (motor or ATPase), or cargo
specificity. The association of a kinase with dynein which we have
observed may allow for modification of not only the intermediate chains
of dynein but also the heavy and light chains, as well as other
accessory proteins such as dynactin. While the functional implications
of this CKII-dynein association remain to be explored, the observation
that CKII can bind to and phosphorylate cytoplasmic dynein should be a
significant step toward understanding the regulation of dynein function
within the cell.
We thank Dr. J. Leszyk for peptide sequencing
work, C. Echeverri and R. Vallee of the Worcester Foundation for
Biomedical Research for their gift of p50 antibody, and E. Holleran, G. Gries, and J. Perkel for reading the manuscript.
Cell and Molecular Biology Graduate Group,
subunit of casein kinase II, a highly conserved
serine/threonine kinase. Affinity chromatography using a dynein column
indicates that both the
and
subunits of casein kinase II are
retained by the column from rat brain cytosol. Although dynactin is
also bound to the column, casein kinase II is not a dynactin subunit. Casein kinase II does not co-immunoprecipitate with dynactin, and it
binds to a dynein intermediate chain column which has been preblocked
with excess p150Glued, a treatment that inhibits the binding of
dynactin from cytosol. Bacterially expressed and purified rat dynein
intermediate chain can be phosphorylated by casein kinase II in
vitro. Further, native cytoplasmic dynein purified from rat brain
can also be phosphorylated by casein kinase II in vitro. We
propose that CKII may be involved in the regulation of dynein function
possibly by altering its cargo specificity or its ability to interact
with dynactin.
2
2 or
2
heterotetramer where the
subunits (molecular mass,
= 42-44
kDa;
= 38 kDa) are catalytic and the
subunits (molecular mass = 26 kDa) are regulatory (for reviews, see Refs. 8-11).
Substrates of CKII include cytoskeletal proteins such as myosin heavy
and light chains, troponin-T,
-tubulin, tau, and MAP-1B (8). CKII function has been implicated in processes of cell cycle regulation, cell division, and signal transduction (12, 13). Although the majority
of cellular CKII localizes to the nucleus, antibodies directed against
CKII also localize to the centrosome (14). Immunolocalization studies
indicate that a population of cellular CKII is associated with the
spindle apparatus in dividing cells (14, 15). Microinjection studies
have also suggested a role for CKII in the regulation of neurite
outgrowth, as the microinjection of antisense oligonucleotides to
CKII
blocked neuritogenesis (16). These studies suggest that CKII
may play a role both in regulating the cell cycle and in neurite
extension; cytoplasmic dynein is thought to be involved in each of
these processes.
Preparation of Cytosol and Purification of Proteins
chain antibody (rabbit polyclonal; Upstate
Biotechnology, Inc., Lake Placid, NY) or anti-CKII
chain antibody
(mouse monoclonal; Transduction Laboratories, Lexington, KY).
antibodies.
-32P]ATP (DuPont NEN), 1000 units of CKII (human
recombinant, New England Biolabs), and 1 × CKII buffer were
mixed, and the final volume brought to 20 µl. An additional tube
containing no CKII was also included to serve as a control. Reactions
were terminated by addition of 5 × SDS sample buffer and boiling.
For in vitro phosphorylation of native cytoplasmic dynein 10 µg of protein (lambda phosphatase-treated or untreated) was mixed
with 250 µM Mg-ATP, 40 µCi [
-32P]ATP,
+/
1000 units of CKII, and 1 × CKII buffer, and reacted at
30 °C. The phosphorylated samples were resolved on 8% or 10% SDS-PAGE as noted, and the dried gels were exposed to x-ray film. The
extent of phosphate incorporation was calculated by excising the gel
bands and counting the radioactivity using a scintillation counter.
Protein concentration in the band was determined by densitometric analysis of the Coomassie-stained gel, using BSA as a standard.
Casein Kinase II Binds to Dynein in Vitro
subunit of casein kinase II from
rat (19) as shown in Fig. 1B.
Fig. 1.
Polypeptides retained by a dynein affinity
column and the comparison of a peptide sequence from p27 with the
predicted sequence of rat CKII. A, DIC eluate was
concentrated and loaded on a 5-20% sucrose gradient. Fractions
corresponding to ~20 S were pooled, methanol precipitated, resolved
by SDS-PAGE, transferred to Immobilon, and Coomassie-stained. This gel
lane shows all the reported subunits of dynactin. The band
corresponding to 27 kDa was excised, subjected to tryptic digest, and
subsequently microsequenced. B, the upper line
shows the sequence of the peptide from p27, and the lower
line shows matching sequence from rat CKII
(19). The
arrows indicate the predicted cleavage sites for
trypsin.
[View Larger Version of this Image (21K GIF file)]
chains and 2
chains in all
higher eukaryotes, we verified that both
and
chains are
retained on a dynein affinity column as shown in Fig. 2.
Rat brain cytosol was loaded on a dynein column, which was then washed extensively and eluted with 1 M NaCl. The resulting
fractions were resolved by SDS-PAGE, transferred onto Immobilon-P,
Coomassie-stained for total protein (Fig. 2A) and
subsequently probed for CKII
and
chain using rabbit polyclonal
and mouse monoclonal antibodies specific for these polypeptides,
respectively (Fig. 2B). As shown in Fig. 2, the immunoblot
indicated that the cytosol contains CKII (lane 1), which
when loaded on to the dynein affinity column (lanes 2-4)
binds to dynein. Not all CKII binds to dynein as indicated by the
presence of CKII
in the flow-through samples (lanes 2 and
5); this may suggest that the column capacity is exceeded or
that there is a pool of cytosolic CKII that does not bind to dynein.
The 1 M NaCl eluate of the dynein column (lane
4) contains both
and
chains of CKII suggesting that intact
CKII holoenzyme binds to the dynein column. A BSA control column which
was constructed identically did not retain any CKII from cytosol
(lanes 5-7). While there was no detectable
chain in the
cytosol as compared to
chain, the
chain is clearly visible in
the salt eluate of the dynein column suggesting that the eluate
fraction is highly enriched in CKII. Difficulty in detecting the
subunit of CKII in the total cytosol may be due to weaker
immunoreactivity of the
antibody. Alternatively, there may be a
lower levels of
subunits present in the cytosol (Fig.
2B, lane 1). Some studies support the hypothesis
that there is a limited pool of CKII
relative to
subunits (35),
and in some species,
subunits are completely lacking (36, 37).
Fig. 2.
CKII and
subunits bind to the dynein
affinity column. Rat brain cytosol (lane 1) was loaded
on a column of DIC (lanes 2-4) or BSA (lanes
5-7). The columns were washed and then eluted with 1.0 M NaCl. The fractions were resolved by SDS-PAGE followed by
transfer onto Immobilon and stained with Coomassie Brilliant Blue
(A) and subsequently probed with antibodies to CKII
and
CKII
(B). Lane 1, cytosol-loaded; lanes
2 and 5, flow-throughs; lanes 3 and
6, final wash; lanes 4 and 7, elution
with 1 M NaCl.
[View Larger Version of this Image (60K GIF file)]
polypeptide might be a subunit of dynactin, we
performed immunoprecipitation using an affinity-purified
anti-p150Glued antibody (Fig. 3). The
immunoprecipitate was observed to contain the dynactin subunits
p150Glued, p50 (dynamitin), and centractin (Arp1) (lane
3), but not CKII (lane 3). We also fractionated the 1 M affinity column eluate by sucrose gradient. We noted that
although a small fraction of CKII did co-sediment with dynactin at
higher 20 S values as was initially observed (see Fig. 1), the majority
of CKII was observed to sediment at low S values closer to the top of
the gradient (data not shown). To verify further that CKII does not
bind to the dynein column as a part of the dynactin complex (5) we pretreated the dynein column with a 2.5 molar excess of a bacterially expressed and purified fragment (residues 152-811) of
p150Glued before loading the brain cytosol onto the column
(Fig. 4). A control column was pretreated with a 10 molar excess of BSA. If CKII is part of dynactin, then the pretreatment
of the DIC column with p150Glued should block CKII binding to
the dynein affinity matrix. When the salt eluates from
p150Glued-treated or BSA-treated DIC affinity columns were
probed for CKII
(Fig. 4B) and centractin (the most
abundant subunit of dynactin; Fig. 4B), high levels of CKII
were found to bind to both columns (Fig. 4B, lanes
4 and 7), whereas centractin bound only to the BSA-blocked column (Fig. 4B, lane 4).
Densitometric analysis of lanes 4 and 7 indicates
that 80% of CKII bound to the p150Glued-blocked dynein column,
while only 2% of centractin bound to such a column compared to the
unblocked dynein column. This result, along with the
immunoprecipitation results described above, suggests that CKII
is
not the reported 27-kDa dynactin subunit. This result also suggests
that CKII may be binding to dynein either directly or indirectly via an
as yet unknown mediator.
Fig. 3.
CKII does not co-precipitate with
dynactin. High speed supernatant of brain cytosol (lane
1) was immunoprecipitated with p150Glued antibody
(lane 3) or control Sepharose beads (lane 2), the
immunoprecipitate resolved on SDS-PAGE, transferred onto Immobilon-P,
analyzed by Coomassie staining (A), and subsequently probed
for p150Glued, p50, centractin, and CKII (B).
While all subunits of dynactin are present in both immunoprecipitate
and column elution, CKII is present only in the DIC eluate. Lane
1, total cytosol used for immunoprecipitation; lane 2,
control immunoprecipitation using Sepharose beads alone without
antibody; lane 3, immunoprecipitate using p150Glued
antibody; lane 4, elution from a dynein column for
comparison.
[View Larger Version of this Image (50K GIF file)]
Fig. 4.
CKII binds to a dynein column independently
from dynactin. Rat brain cytosol (lane 1) was loaded on
intermediate chain column that was either pretreated with BSA
(lanes 2-4) or a p150Glued fragment (lanes
5-7). The columns were washed and eluted with 1.0 M
NaCl and fractions resolved by SDS-PAGE, transferred onto Immobilon-P,
Coomassie-stained (A), and subsequently probed for CKII
or centractin (B) as indicated. Results show that while dynactin was blocked by p150Glued pretreatment, CKII was not.
Lane 1, cytosol input; lanes 2 and 5,
flow throughs; lanes 3 and 6, final wash;
lanes 4 and 7, elution with 1 M
NaCl.
[View Larger Version of this Image (67K GIF file)]
-32P]ATP. The resulting time course shown in Fig.
5A indicates that dynein is readily
phosphorylated by purified CKII (lanes 2-7). Incorporation
of phosphate into the dynein intermediate chain in this experiment
approached 2 mol of phosphate per mol of polypeptide (Fig.
5C). This suggests that there are at least 2 CKII
phosphorylation sites in DIC.
Fig. 5.
Phosphorylation of dynein by CKII in
vitro. A, bacterially expressed and purified
intermediate chain was subjected to CKII phosphorylation in
vitro for the following time intervals, lane 2, 5 min;
lane 3, 10 min; lane 4, 15 min; lane
5, 30 min; lane 6, 60 min; lane 7, 120 min.
Lane 1 is a 15-min control sample lacking CKII. The
reactions were stopped by adding 5 × SDS sample buffer to the
reaction tubes and boiling before analysis by SDS-PAGE followed by
autoradiography. The bands corresponding to polypeptides of 27 and 24 kDa are amino-terminal fragments of the dynein intermediate chain.
B, purified dynein from rat brain cytosol was subjected to
in vitro phosphorylation by CKII with (lanes
1-3) or without (lanes 4-6) lambda protein
phosphatase pretreatment. Control samples (lanes 7-9) did
not contain any CKII. The reactions were stopped at 45 min (lanes
1, 4, and 7), 75 min (lanes 2,
5, and 8), and 120 min (lanes 3,
6, and 9). The subsequent analysis was done as
described in A. The results show that dynein is
phosphorylated by CKII and phosphatase treatment enhances the amount of
radiolabeled ATP incorporation. C, graph depicts moles of
phosphate incorporated into a mole of recombinant DIC in
vitro by CKII. This experiment was done separately from
A and indicates that, at saturation, approximately 2 phosphate moles are incorporated per DIC molecule.
[View Larger Version of this Image (27K GIF file)]
and
subunits of CKII bind
to a dynein affinity column (Fig. 1) and that the first 120 amino acids
of the DIC are sufficient for CKII binding (data not shown).
subunit (molecular mass = 26 kDa) matches
closely with the reported 27-kDa subunit of the dynactin complex (20).
Thus there was the possibility that CKII
may represent this as yet
uncharacterized dynactin subunit since it is known that dynactin
complex binds to the intermediate chain column. We tested this
possibility by immunoprecipitation as well as in column blocking
experiments. The results clearly demonstrate that CKII
cannot be a
dynactin component since it does not co-precipitate with other dynactin
subunits nor is its binding to dynein affected by pretreatment of the
column with p150Glued. The pretreatment of the dynein column
with excess p150Glued blocked subsequent binding of dynactin as
evidenced by lack of retention of centractin (Arp1; Fig. 4, lane
4) on such a column. This result establishes that association of
CKII with dynein is independent of the dynein-dynactin interaction.
However, we have not yet determined whether the binding of CKII to
dynein is direct or is indirect and mediated by an as yet unidentified
factor. It also remains to be determined if there is a true 27-kDa
subunit of dynactin or whether contaminating CKII
subunit was taken
to be a dynactin subunit (20). Using affinity chromatography and immunoprecipitation assays, we have recently demonstrated that actin is
not a stoichiometric component of dynactin (7), as was suggested in an
earlier report (20).
*
This work was supported in part by National Institutes of
Health Grant GM48661 (to E. L. F. H.) and by a Predoctoral
Fellowship from the American Heart Association, Southeast Pennsylvania
Affiliate (to S. K.). 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.
¶
Recipient of an Established Investigatorship from the American
Heart Association during the tenure of this work. To whom
correspondence should be addressed: University of Pennsylvania, 143 Rosenthal Bldg., 3800 Spruce St., Philadelphia, PA 19104-6048. Tel.:
215-573-3257; Fax: 215-898-9923; E-mail:
holzbaur{at}phl.vet.upenn.edu.
1
The abbreviations used are: DIC, dynein
intermediate chain; CKII, casein kinase II; BSA, bovine serum albumin;
PAGE, polyacrylamide gel electrophoresis; PIPES,
1,4-piperazinediethanesulfonic acid.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.