(Received for publication, July 13, 1995; and in revised form, July 25, 1995)
From the
A Ca-calmodulin-activated histone 3 kinase was
partially purified from nuclear extracts of dividing and quiescent rat
heart endothelial cells. The histone 3 phosphorylating activity was
20-100-fold higher in quiescent than in dividing cells. Base
hydrolysis followed by amino acid analysis revealed that histone 3 was
phosphorylated on arginine. Further investigations were conducted to
determine whether phosphorylation of histone 3 also occurred in
vivo. Cells were incubated for 3 h in a phosphate-free medium
supplemented with [
P]phosphoric acid. It was
observed that the nuclear content of arginine-phosphorylated histone 3
was considerably higher in quiescent than in dividing rat heart
endothelial cells. The histone 3 arginine kinase is a component of a
complex containing a Ca
-dependent calmodulin-binding
protein of apparent molecular mass of 85 kDa. Using polyclonal
antibodies to an 85-kDa protein, also the major
Ca
-dependent calmodulin-binding component of the
histone 3 arginine kinase from calf thymus, an immunoreactive protein
of identical apparent molecular mass was found to be present in equal
amounts both in dividing and quiescent cells. We propose that the
85-kDa protein is either the histone 3 arginine kinase or one of its
subunits and that phosphorylation of histone 3 is involved with cell
cycle exit in eukaryotes.
Protein phosphorylation/dephosphorylation plays an important
role in a number of cellular activities in eukaryotes (reviewed in (1) ). Such activities include initiation of mitosis (reviewed
in (2) ) and regulation of transcription (reviewed in (3) ). Among the proteins phosphorylated are transcription
factors(4, 5) , topoisomerase II(6) , and
histones 1 and 3(7, 8) . Phosphorylation of histone 3
(H3) ()is associated with mitosis(7, 9, 10) and is suggested to be closely associated with chromatin
condensation (11) . Stimulation of quiescent cells by growth
factors, phorbol esters, okadiac acid, and protein synthesis inhibitors
also leads to the rapid phosphorylation of H3(12) . Mahadevan et al.(12) suggested that the rapid phosphorylation
of H3 modulates nucleosomal characteristics and potentially regulates
early gene expression at the structural level. The phosphorylation
sites of H3 in all of the above reports were shown to be in the
N-terminal region of H3, on serine or threonine residues. Our knowledge
of the kinases that phosphorylate H3 is limited to a kinase identified
as a component of the HeLa cell(13) , a 38-kDa chromatin-bound
H3 kinase from calf thymus chromatin(14, 15) , the
catalytic subunit of cAMP-dependent kinase used by Shibata et
al.(11) , and a chromatin-bound
Ca
-CaM-activated kinase from calf thymus (16) and from mouse leukemia cells(17) . The kinase
from mouse leukemia cells is the first to phosphorylate a basic amino
acid (arginine) in a Ca
-CaM-dependent
manner(17) . Interestingly, three of the four phosphorylated
arginine residues in H3 are within the 15-amino acid C-terminal tail of
H3. Such a modification in the C terminus of the protein will alter the
overall charge of that region and potentially alter the binding of H3
to DNA during nucleosome assembly/disassembly. The present report is
intended to demonstrate that the Ca
-CaM-dependent
arginine phosphorylation of H3 is correlated with cell cycle exit in
rat heart endothelial (RHE) cells and that the H3 kinase is a regulated
kinase, potentially a component of a cascade responsible for cell cycle
exit.
CaM affinity-purified fractions from RHE cells were assayed
for their histone phosphorylating activities (Fig. 1). The
reaction mixtures contained 100 µg of total histones and 2 µg
of CaM affinity-purified fractions per assay. Equal amounts of histones
(15 µg) were loaded per lane. As can be observed from Fig. 1, dividing cells contained a considerably lower level of
H3 phosphorylating activity compared to the activity from quiescent
cells (Fig. 1, lanes 1 and 3, respectively).
The presence of Ca-CaM in the assay did not
noticeably activate the H3 kinase from dividing cells (Fig. 1, lane 2), but activated the kinase from quiescent cells by a
factor of at least 10 times (Fig. 1, lane 4). The band
labeled H3 (deg.) has been identified as H3, lacking its
N-terminal 27 amino acids, which is also phosphorylated by the
Ca
-CaM kinase from quiescent cells. In fact, the
presence of H3 (deg.) in the assay mixture helped us exclude the N
terminus from being the primary site of H3 phosphorylation. The
phosphorylated amino acid in H3 was identified as arginine. Briefly,
P-labeled H3 was hydrolyzed for 3 h in 3 M KOH at
124 °C. Samples were cooled on ice, and equimolar amounts of
perchloric acid were added in order to precipitate salts and to leave
the amino acids in solution. Samples were then centrifuged, and the
supernatants were dried. Amino acids were derivatized with
phenylisothiocyanate and analyzed according to Wakim and
Aswad(17) . It was observed that over 90% of the
phosphate-labeled amino acids migrated with identical retention times
as that of phosphoarginine (data shown below). This is in agreement
with earlier observations from our laboratory showing that H3 is
phosphorylated primarily on three arginine residues present in its
C-terminal tail(17) . Also shown in Fig. 1is the
presence of a Ca
-CaM-dependent phosphoprotein with an
apparent molecular mass of 50 kDa. The identity of this protein has not
been determined. It is clear from Fig. 1that quiescent cells
contain at least 20-100-fold more of the
Ca
-CaM-dependent H3 phosphorylating activity than do
dividing cells.
Figure 1:
Autoradiogram of
phosphorylated histones in the absence (lanes 1 and 3) and presence of CaM (lanes 2 and 4).
Phosphorylation was performed in the presence of 100 µg of calf
thymus histones per assay reaction and 2 µg of CaM
affinity-purified fractions from dividing (lanes 1 and 2) and quiescent (lanes 3 and 4) cells. 15
µg of total histones were loaded per lane. H3 is the predominant
protein phosphorylated in the presence of the CaM affinity-purified
fraction from quiescent cells (lane 3). The presence of
Ca-CaM increased the phosphorylation of H3 by at
least 10-fold (lane 4).
To further pursue the possibility that arginine
phosphorylation of H3 is related to cell quiescence it was important to
demonstrate differences in the levels of arginine phosphorylated H3 in vivo between dividing and quiescent cells. RHE cells were
collected either while dividing, after reaching confluency followed by
a 2-day serum deprivation, or after reaching confluency then replating
in the presence of serum to obtain dividing cells. As can be seen from Fig. 2, approximately 25% of the cells were in transit through
the S phase (Panels A and C) while only a minimum of
2.6% of the cells grown to confluency and with serum deprivation were
in the S phase (Panel B). Under confluent conditions the
majority (over 90%) of the cells were arrested within the G phase of the cell cycle. In parallel, in vivo incorporation of
P into H3 was also determined in
dividing and confluent cells. As described under ``Experimental
Procedures,'' cells were incubated in the presence of
[
-
P]phosphoric acid for 3 h. Nuclear
extracts were prepared, and proteins were separated onto two identical
16% SDS-polyacrylamide gels. Equal amounts of protein were loaded per
lane. Both gels were transferred to PVDF membranes. One blot was
treated with 10% acetic acid and the other with 0.5 M KOH for
3 h at 50 °C. Fig. 3represents an autoradiogram of the acid (A) and base (B) treated blots. It is clear from this
figure that, in the case of quiescent cells, a phosphorylated H3 band
was observed in the blot that was treated with KOH (Fig. 3B, lane 2). The same band was not
observed in the blot treated with acid (Fig. 3A, lane 2). This indicated that in the case of quiescent RHE
cells a basic amino acid was phosphorylated in H3. In the case of
dividing cells, the autoradiograph showed that the same phosphorylated
band was absent from both acid- and base-treated blots (Fig. 3, A and B, lanes 1 and 3). The
phosphorylated amino acid in H3 was further identified to be arginine (Fig. 4). The H3 band from quiescent cells was excised from the
blot and base-hydrolyzed followed by identification of the
phosphorylated amino acid. As can be observed from Fig. 4, about
90% of the
P counts migrated with an identical retention
time as phosphoarginine. This strongly indicates that the principle
phosphorylated amino acid is arginine.
Figure 2: The distribution of cells through the cell cycle was carried out using a FACStar plus flow cytometer. Collected data were processed by the computer program Modfit 5.02. Panel A represents cells in exponential growth. Panel B represents the same cell population grown to confluency and serum-deprived for 2 days. Panel C represents replated confluent cells that were allowed to grow in the presence of serum.
Figure 3:
Autoradiogram of phosphorylated proteins
from quiescent and dividing cells. Cells were incubated in the presence
of [-
P]phosphoric acid. Nuclear extracts
were prepared and separated on two 16% SDS-polyacrylamide gels.
Proteins were transferred to PVDF membranes as described under
``Experimental Procedures.'' Blots were treated either with
10% acetic acid (A) or with 0.5 M KOH (B) at
50 °C for 3 h. Lanes A1 and B1 represent nuclear
extracts from dividing cells, lanes A2 and B2 from
quiescent cells, and lanes A3 and B3 from dividing
cells that were grown to quiescence then transferred to new plates and
allowed to grow in the presence of fetal calf serum. For the course of
the
P incorporation studies (3 h) cells were grown in the
absence of fetal bovine calf serum and in the presence of 100 ng/ml
insulin-like growth factor-I, 50 ng/ml
-fibroblast growth factor,
and 12.5 units/ml heparin. In the case of quiescent cells,
phosphorylation of H3 was acid-labile. Acid treatment (lane
A2) removed the phosphate from H3, while base treatment (lane
B2) did not.
Figure 4:
Reversed phase HPLC of base hydrolyzed and
phenylisothiocyanate-derivatized amino acids. Phosphoarginine standard (solid line), elution profile of radiolabeled phosphoamino
acids from in vitro phosphorylated H3 (- - - -
),
and from in vivo phosphorylated H3 from quiescent RHE cells
(
- - - -
).
Polyclonal antibodies to the 85-kDa protein from calf thymus were raised in rabbits and used to identify immunoreactive proteins present in RHE cell extracts. It was observed that an immunoreactive protein with 85-kDa apparent molecular mass was present in both dividing and quiescent cells (Fig. 5B, lanes 1 and 2, respectively). Silver staining of CaM affinity-purified fractions from dividing and quiescent cells (Fig. 5A, lanes 1 and 2, respectively) showed very similar protein patterns. The patterns of immunoprecipitated proteins from dividing and quiescent RHE cells were also very similar (Fig. 5A, lanes 3 and 4, respectively). As can be observed from Fig. 5B, lanes 1 and 2, the 85-kDa immunoreactive protein was detected at a nearly equivalent amount by immunoprecipitation of proteins from both dividing and quiescent cells. The intense band below the 85-kDa protein represents the IgG heavy chain. The data presented thus far demonstrate that phosphorylation of arginine in H3 potentially plays a role in cell cycle exit and that the kinase responsible for its phosphorylation, while present in nearly equal amounts in both dividing and quiescent cells, is activated 20-100-fold in quiescent cells.
Figure 5:
Silver-stained CaM affinity-purified
nuclear fractions (Panel A, lanes 1 and 2)
and immunoprecipitated cellular fractions (Panel A, lanes
3 and 4) from dividing and quiescent RHE cells,
respectively. Panel B represents the identification of
P85 immunoreactive proteins present in dividing and quiescent
cells (Panel B, lanes 1 and 2,
respectively). It can be observed from this figure that the 85-kDa
protein is present at equivalent amounts in both dividing and quiescent
cells. The strong band below the 85-kDa protein represents the IgG
heavy chain.
The H3 arginine
phosphorylating activity consisted of a protein-DNA complex and no
detectable RNA. The ratio of protein to DNA was 95:5%, w/w. The complex
contained a Ca-CaM-binding protein of 85-kDa apparent
molecular mass (Fig. 6). Lane 1 represents silver
staining of the CaM affinity-purified complex after protein separation
on a 12% SDS-polyacrylamide gel. Lane 2 represents the binding
of biotinylated CaM to an identical sample after blotting to
nitrocellulose. As can be observed from lane 2, a protein with
an 85-kDa apparent molecular mass bound CaM in a
Ca
-dependent manner. The 85-kDa protein from RHE is
believed to be either identical or homologous to the 85-kDa CaM-binding
protein isolated from calf thymus (16) . Both proteins have the
same apparent molecular mass, were present in the CaM affinity fraction
purified under identical conditions, and bound CaM in a
Ca
-dependent manner. We propose that the 85-kDa
protein is either the H3 arginine kinase or one of its subunits and
that phosphorylation of H3 is involved with cell cycle exit in
eukaryotes.
Figure 6:
Identification of
Ca-CaM-binding protein in CaM affinity-purified
nuclear fractions from quiescent RHE cells. Lane 1 represents
the silver-stained fraction. Lane 2 represents a Western blot
of an identical fraction to that in lane 1. Using biotinylated
CaM the 85-kDa protein was the only protein shown to bind
CaM.
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