(Received for publication, October 2, 1995; and in revised form, December 13, 1995)
From the
We describe the isolation and characterization of cDNAs encoding
full-length human and murine cyclin G1 and a novel human homologue of
this cyclin designated cyclin G2. Cyclin G1 is expressed at high levels
in skeletal muscle, ovary, and kidney. Following an initial
up-regulation from early G to G
/S phase, cyclin
G1 mRNA is constitutively expressed throughout the cell cycle in T and
B cell lines. In contrast, in stimulated peripheral T cells, cyclin G1
mRNA is maximal in early G
phase and declines in cell cycle
progression. Cyclin G1 levels parallel p53 expression in murine B
lymphocytes; however, in several human Burkitt's lymphomas,
murine lymphocytes treated with transforming growth factor-
, early
murine embryos, and several tissues of p53 null mice, cyclin G1 levels
are either inverse of p53 levels or expressed independent of p53. The
cyclin G1 homologue, cyclin G2, exhibits 60% nucleotide sequence
identity and 53% amino acid sequence identity with cyclin G1, and like
cyclin G1, exhibits closest sequence identity to the cyclin A family.
Distinct from cyclin G1, the amino acid sequence for cyclin G2 shows a
PEST-rich sequence and a potential Shc PTB binding site. Cyclin G2 mRNA
is differentially expressed compared to cyclin G1, the highest
transcript levels seen in cerebellum, thymus, spleen, prostate, and
kidney. In contrast to the constitutive expression of cyclin G1 in
lymphocytes, cyclin G2 mRNA appears to oscillate through the cell cycle
with peak expression in late S phase.
Transitions through the eukaryotic cell division cycle are
primarily coordinated by the sequential activation of cyclin-dependent
kinases (CDKs) ()which are, in turn, regulated by subunit
associations and phosphorylation (reviewed in (1, 2, 3) ). The cyclins represent a group of
closely related molecules which primarily function at specific stages
of the cell cycle as regulators of CDK activity by binding and forming
active complexes with specific partner CDKs. This cyclin-CDK
association is in part determined by the conserved cyclin region of
110 amino acids referred to as the cyclin
box(4, 5, 6) . The cyclin box exhibits
30-50% identity between the different cyclin types, the
consensus sequence varying depending on the class and subclass of
cyclin(5, 7, 8) . Cyclins have been
classified into different groups on the basis of their structural
similarity, functional period in the cell division cycle and regulated
expression. In addition to providing positive growth control, CDKs and
cyclin-CDK pairs may participate in metabolism and signal transduction
unrelated to cell cycle as evidenced by pho80-pho85 cyclin-CDK complex
participation in yeast phosphate metabolism(9, 10) ,
the expression of CDK5 in nonproliferating brain
tissue(11, 12, 13) , and the SRB10/11
cyclin-CDK regulator of RNA polymerase II(14) .
To date at
least 12 different cyclins in budding yeast, 4 in fission yeast, and 10
in mammalian cells (cyclins A-H with multiple family members for some
types) are known, all primarily displaying sequence homology within the
cyclin box
region(1, 2, 3, 4, 5) . Two
broad classes of cyclins, based on genetic complementation of cell
cycle mutations, have been identified in yeast: the G or
``START'' cyclins regulating G
to G
to S phase transition, and the G
/M phase class
necessary for mitosis. In higher eukaryotes, the G
cyclins
(reviewed in Refs. 2, 15, and 16) are represented by the D-type
(D1-D3)(12, 17, 18, 19, 20, 21) and
cyclins C (12, 22) and E(12, 23) ;
the mitotic cyclins by cyclin A (13, 24) and the
B-type (B1-B3)(25, 26, 27) , with cyclin A
acting earlier than the B-type. Cyclin F(28) , as well as
cyclin A (when complexed with CDK2), are believed to function during S
phase. Cyclin H is reported to act indirectly at S to G
transition by regulating, in a complex with the CDK activating
kinase MO15, the phosphorylation of other cyclin-CDK complexes in a
cyclin/kinase signal transduction cascade(29, 30) .
Although partner CDKs have been identified for many cyclins, a number
of CDK related molecules have been discovered for which no cognate
cyclin is known (reviewed in Refs. 2, 3, and 31). The activity of some
cyclin-CDK complexes is also subject to regulation through interaction
with CDK inhibitors (p15 and p16
,
p21
, and p27
) produced in response
to negative stimuli to prevent cell cycle progression (reviewed in (32, 33, 34) ).
Cyclin G was first
identified serendipitously in screens for src family kinases in rat
fibroblasts(35) , and later by differential screen for
transcriptional targets of p53(36) . The function of cyclin G
has not been determined, however, identification of multiple p53
binding sites in the genomic DNA(37) , transactivation by
wild-type p53, and induction following -irradiation (responses
associated with checkpoint and cell cycle
arrest(38, 39) ) implicate cyclin G in negative growth
control or DNA damage repair(36, 37) . The absence of
either prototypic protein destabilizing (PEST) sequences of G
class cyclins(40, 41) , or the
``destruction box'' sequence controlling the
ubiquitin-dependent degradation of mitotic cyclins(42) ,
indicate alternate regulation of cyclin G protein expression.
Additionally, the presence of an EGF-R/ErbB-like autophosphorylation
motif has suggested a role for cyclin G in signal
transduction(35) . We have isolated cDNAs encoding full-length
human and murine cyclin G which we have designated cyclin G1. Here we
describe the expression pattern for cyclin G1 mRNA throughout the cell
cycle in B and T cells, and provide correlative evidence for its
regulation by p53 in murine B lymphocytes, yet contrast this with mRNA
expression inverse to p53 levels during development and independent of
p53 in various cell lines and differentiated tissues. In parallel, we
describe the cloning of a novel human cyclin G1 homologue, designated
cyclin G2, and compare its tissue and cell cycle position-specific
transcript expression to that of cyclin G1. The presence of two
potentially phosphorylated tyrosines in the G2 protein sequence distal
to CDK interaction may implicate cyclin G2 as a component of signal
transduction pathways regulating cell growth.
For human cyclin G1, three fragments overlapping
different regions of the GeneBank sequence for rat cyclin G1 (EMBL
Z24820, X77794, and GB T16966) were used to generate a composite,
putative full-length human cyclin G1 sequence and design corresponding
oligonucleotide primers (forward primers: 5`-AAGATGATAGAGGTACTG-3`,
5`-AAGATGATAGAGGTACTGACAACAACTGACTC-3`, and 5`-CAGGAGTCTAGATGTCAGCC-3`;
reverse primers: 5`-CAGTTAAGGGACCATTTCAGGAATTGTTGG-3`, and
5`-GCAATGATAGACAATGCCAA-3`) to generate PCR fragments with partial and
full ORFs. These fragments were cloned into the pCRII
cloning vector following the TA Cloning
kit protocol
(Invitrogen Corp.), their sequence, determined as described below, was
subsequently utilized to generate probes for human and murine cDNA
libraries. For murine cyclin G1, original forward primers
(5`-GGCGGATCCAAGATGATAGAAGTACTG-3`,
5`-GGCGGATCCCTGAGTCTAACTCAGTTCTTTGGC-3`) and reverse primers:
(5`-GGCGAATTCATTCTACTCCTTCTGTTAACTCCAC-3`, and
5`-GGCCTTAAGCTAACCCATGGTTTCGGGAATTGTTGGG-3`) were designed (with
engineered restriction enzyme recognition sites at the termini) from
regions showing nearly 100% identity in nucleotide sequence alignment
between the GeneBank rat cyclin G1 sequence (EMBL X70871) and our
composite putative human cyclin G1 sequence. When differences or
ambiguities were present between the sequences in the alignment, the
nucleotides of the rat sequence were used to construct the primers for
the murine cyclin G1. These primers were then utilized to generate PCR
fragments from murine splenocyte and peripheral blood leukocyte cDNA
libraries (Clontech, Palo Alto, CA) that were then cloned and sequenced
as described below. Sequences of PCR-derived fragments were compared to
those of cDNA library clones obtained by hybridization screening
(described below).
For human cyclin G2 an automated data base search
using BLASTX was instituted to search the translation products from all
six reading frames of the GenEMBL sequence updates on a weekly basis
and compare the deduced amino acid sequences to various regions of rat
cyclin G1, in particular the cyclin box. One such search identified a
361-bp EST sequence obtained from the cDNA derived from brain tissue of
a human infant (EMBL Z46163). This EST showed a BLAST probability (p) value of 2.1e to the first 44 amino
acids of the rat cyclin G1 box used for the search. Subsequent BLASTX
translation of the entire EST sequence yielded p values
= 3.3e
, 5.6e
, and
1.1e-
to the respective deduced amino acid sequence of
human, rat, and mouse cyclin G1 sequences now present in the GenEMBL
data base. The deduced amino acid sequence identities ranged between 66
and 44% for two respective contiguous regions in two different reading
frames, which together encompassed the entire EST sequence.
Oligonucleotides were designed to the 5` and 3` termini of this EST
(forward primers: 5`-GGGGTCCAACTTCTCGGGTTGTTGAACG-3` and
5`-CCAACTTCTCGGGTTGTTGAACGTCTACC; reverse primers:
5`-GTACATTTACACTGACTAATCCGG-3` and 5`-CTAATCCGGATCACATCATGAGTG-3`) and
used to PCR clone this fragment from a human brain cDNA library and
cDNAs prepared from human B and T cell lines. These fragments were
isolated, cloned, and sequenced as described above and below. The
sequence-confirmed and corrected clones encompassed a single,
contiguous ORF encoding a peptide with 53% amino acid identity to human
cyclin G1. This cDNA fragment was then labeled with digoxigenin and
used to probe a Jurkat cDNA library as described below.
To determine the sequence for the 3` end of the cyclin G2 ORF not encompassed by the cDNA library clones, overlapping cDNAs from both Jurkat and Daudi total RNA were obtained using the PCR based 3`-RACE method and reagents (Life Technologies, Inc.). Following the manufacturer's suggested protocol, first strand synthesis of the cDNA template was obtained with a supplied tagged oligo(dT) primer. Second strand synthesis was done with two different G2 specific forward primers in the 3` exons on either side of the 3` most splice-site junction (5`-GGACAACAGCTACTATAGTGTTCC-3` and 5`-GAAAGTGAGGACTCTTGTG-3`) and a supplied 3` primer annealed to the template cDNA at 65 °C in a 20-cycle PCR. A second round of PCR with the nested 5` primers was subsequently performed for 15 cycles on an aliquot of cDNA obtained from the above 2nd strand synthesis. The products were TA cloned and multiple independent clones were isolated and sequenced (described above and below).
To PCR clone a mature cDNA encompassing the cyclin G2 ORF, oligonucleotides were designed (with appended restriction enzyme sites) to sites 5` to initiation codon and 3` to the last intron/exon junction near the termination codon (forward primer: 5`-GGCGGATCCCTCTGTGTGGTGTCTTTACTG-3`; reverse primer: 5`-GCCGAATTCGGTGCACTCTTGATCACTGGG-3`) and used to amplify DNA fragments from Jurkat cDNA as described above.
The glyoxal denaturation of total RNA and electrophoresis in glyoxal-agarose was done following standard protocol(44) . After electrophoresis a control check of the relative amount and quality of the RNA was done by short wave UV fluorescent shadowing of the ribosomal RNAs on a F-254 TLC plate. The fractionated RNAs were transferred and fixed to Pall Biodyne A nylon membranes (Life Technologies, Inc.) followed by removal of residual glyoxal as described(44, 47) .
The
[-
P]dTTP and
[
-
P]dCTP radioactive labeling of DNA
fragments was done using PCR generated and GeneClean II
isolated DNA fragments as templates and reagents obtained from
the Boehringer Mannheim Random Priming kit. Hybridization of DNA probes
to Northern blots was done essentially following the method
``B'' protocol described by Pall BioSupport. PhosphorImaging
(Molecular Dynamics) exposure of the washed Northern blot filters was
routinely done immediately prior to autoradiography.
Hybridization screening of a Jurkat cDNA
library with the PCR-isolated cDNA probes identified 5 independent
human clones comprising two overlapping cDNAs encompassing full and
partial human cyclin G cDNA. Screens of the murine peripheral blood
leukocyte library identified 10 clones comprising four overlapping
partial cDNAs encompassing the full cyclin G ORF. Sequence was verified
by double stranded analysis of all human and murine PCR and cDNA
library cyclin G clones obtained from lymphoid tissues and cell lines.
The cyclin G ORF in both human and murine extended another
132-135 nucleotides, encoding a protein with an
NH-terminal region of 45 (murine) or 46 (human) amino acids
beyond the cyclin box with a revised molecular mass of 34 kDa (Fig. 1). This finding is consistent with the
NH
-terminal flanking region and revised molecular weight
recently reported in the full-length sequence of rat cyclin
G(37) . Here we designate this cyclin G1 to distinguish it from
its homologue. In the human 3`-UTR we find base substitutions of a
thymidine for a guanosine, an adenosine for a guanosine, and a
guanosine for an adenosine at 208, 263, and 346 nucleotides 3` to the
TAA STOP codon, respectively(48) . Comparative analysis of the
cyclin G1 3`-UTRs determined that the human sequence lacked a block of
800-1050 nucleotides found in the rodent sequences, yet
contains an island of conserved sequence shared by all three 3`-UTRs. A
concentration of multiple copies of the sequence AUUUA, a mRNA
destabilization motif (49, 50, 51) , is found
within this island of conserved sequence (data not shown).
Figure 1:
Sequences of human, mouse, and rat
cyclin G1 gene ORFs. Comparative nucleotide sequence alignment of
mouse, rat, and human cDNAs encoding cyclin G1 is shown with flanking
5` and 3` sequences and the deduced amino acid sequence for the human
sequence. The start (+) position of the cycG1 gene ORF is
defined by the first ATG (bold type shaded dark gray)
following in-frame stop codons (light gray shade), and the end
is determined by the in-frame stop codons 3` to the ATG. The numbering
of nucleotide positions is shown to the right of each sequence
relative to the start codon (+1). Identical nucleotides conserved
in at least two sequences are denoted by uppercase letters.
Nucleotides missing from prior published sequences are boxed in bold underlined type, and a vertical double bar indicates the 5` end of the previously published human cDNA
sequence. The corrected start site predicts a protein with an
additional 45-46 amino acids NH-terminal to the
methionine at the beginning of the cyclin box. The cyclin box region is
indicated by the shaded region bordered by double lines. The
postulated EGF/avian ErbB-like autophosphorylation domain with a
potential tyrosine phosphorylation site is indicated by the dashed-outlined box with shaded identities to either the EGF
or ErbB kinase domain. The potential phosphotyrosine is indicated by an asterisk.
Figure 2:
Size and tissue distribution of cyclin G1
transcript in murine tissues and cell lines. Northern blot analysis of
15 µg of total RNA from murine tissues (left panel)
and cell lines (right panel), probed for cyclin G1 and GAPDH
(as a control for relative amount and quality of mRNA). The position of
RNA standard markers are shown on the left and the 3.4-kb
cyclin G1 mRNA is indicated. Right panel, transcript levels in
B cell (lanes 1-3), T-cell (lanes 4-7),
thymic epithelium (lanes 8-9), monocyte and melanoma (lanes 10 and 11, respectively) cell
lines.
Expression of
murine cyclin G1 through the cell cycle in B lymphocytes was examined
in cell lines from two distinct stages of developmental progression:
WEHI-231, representing an IgM immature B cell which
undergoes growth arrest upon B cell receptor cross-linking, and Bal-17
representing an IgM
IgD
mature B cell
phenotype which continues to proliferate upon IgM
cross-linking(52) . Logarithmically growing cultures of
these two B cell lines were separated into populations at progressive
stages of the cell cycle by centrifugal elutriation, and Northern blot
analysis was performed on total RNA isolated from each population (Fig. 3, A and B). WEHI-231 cyclin G1
expression increased
2.5-fold in cells transiting early G
phase and remained at this level throughout the rest of the cell
cycle. In contrast, cyclin G1 message in corresponding fractions from
Bal-17 was markedly reduced from that seen in WEHI-231
(
5-6-fold less than the maximum WEHI-231 transcript level).
The constitutively low level of cyclin G1 throughout the cell cycle
exhibited no increase upon early G
phase transition (Fig. 3C).
Figure 3:
Northern analysis of cyclin G1 expression
through the cell cycle of two murine B cell lines correlated with p53
expression. A, FACS profile of cellular DNA content from the
elutriated cell fractions for corresponding Northern blots shown in B and D. B, Northern blot analysis of total
RNA from the cell cycle positioned cells of Bal-17 (left) and
WEHI-231 (right) hybridized with cyclin G1, cyclin D2, and
GAPDH P-labeled cDNA probes. The fraction numbers below
each lane correspond to the elutriated fraction in the FACS profiles
shown in A. C, bar graph for comparison of Cyclin G1
expression throughout the cell cycle relative to GAPDH in both Bal-17
and WEHI-231. The y axis indicates the normalized signal as a
percentage of the maximum level detected. D, p53 levels in
Bal-17 and WEHI-231 by examination of protein (upper panel)
and mRNA levels (lower panel). Immunoprecipitation of p53 from
lysates of metabolically labeled EL-4, Bal-17 and WEHI-231 cells (upper panel). Protein A alone pre-clear immunoprecipitate
controls (PC) are compared to two independent p53
immunoprecipitates (IP1 and IP2) produced with two
different commercial antibodies specific for wild-type p53
(``Materials and Methods''). Northern blot for p53 mRNA
levels performed on the same membrane shown in B.
Our earlier survey of wild-type p53 in
various cell lines had indicated that no detectable amount of wild-type
p53 protein was precipitable (using either of two different monoclonal
antibodies to wild-type p53) from metabolically labeled protein lysates
of Bal-17, in contrast to that seen for WEHI-231 and EL-4 (Fig. 3D, top panel). The report of the cyclin G gene
as a target of p53 activation (36) prompted us to further
investigate the status of p53 expression in these two cell lines. Lack
of a detectable wild-type p53 protein in Bal-17 was verified by
Northern blot analysis of both Bal-17 and WEHI-231. Probing the same
membrane used for the cell cycle Northern analysis of cyclin G1, shown
in Fig. 3B, for p53 message clearly indicated that no
p53 transcript, and thus no p53 protein, is present in Bal-17 cells (Fig. 3D, bottom panel). p53 function was confirmed in
WEHI-231 by actinomycin D induced G arrest(53) .
Predictably, Bal-17 cells under the same conditions were nonresponsive
to DNA damage and showed no inhibition of S phase entry (data not
shown). Taken together these results are in agreement with those of
Okamoto and Beach (36) that the cyclin G1 gene in murine
lymphocytes is a target for transcriptional activation by wild-type
p53.
Figure 4:
p53 independent cyclin G1 mRNA expression
in tissues, during embryogenesis, and in p53-negative cells. A,
top: Northern blot analysis of cyclin G1 transcript levels in the
indicated tissues from p53 wild-type (+/+), heterozygous
(+/-), and gene knockout mice (-/-) compared to
the GAPDH and wild-type p53 transcript levels (due to the apparent low
level of p53 in muscle tissues only selected representative samples
probed for p53 are shown). Bottom, quantitation of cyclin G1
mRNA levels relative to GAPDH levels in the tissues examined above. B, Northern blots of murine embryos examined for cyclin G1
mRNA expression relative to wild-type p53 and GAPDH. The stage, in days
of embryonic development, is indicated above each lane. The
RNA and blot on the left was prepared with 15 µg of total
RNA/lane while the blot on the right (Clontech) contains
2.5 µg of Poly(A)
mRNA/lane. Blots were
sequentially probed for cyclin G1, p53, and GAPDH. C,
examination of cyclin G1 expression in Bal-17 and WEHI-231 cells
treated over 24 h (time point above corresponding lane) with the growth
inhibitor TGF-
and compared to GAPDH. PhosphorImager quantitation
of cyclin G1 mRNA relative to GAPDH is shown below
blots.
To begin to investigate the relationship
between p53 and cyclin G1 expression during embryogenesis, comparative
Northern blot analysis was performed on RNA isolated from embryos of
wild-type mice at progressive stages of development. Our initial
comparison of 10- and 16-day embryos indicated cyclin G1 transcript
increased during development in an inverse relationship to p53
expression (Fig. 4B, left panel). This result prompted
examination of RNA from embryos covering a more extensive course of
development (Fig. 4B, right panel). Day 7 is a highly
proliferative stage when the duration of the cell cycle S and G phases are condensed in certain regions of the mouse embryo and
the differentiation commitment of some cell lineages begins with
gastrulization and primitive streak formation (54, 55) . Cyclin G1 message was very high relative to
both GAPDH and p53 at day 7. Between day 7 and day 11 there appeared to
be a switch to considerably reduced cyclin G1 levels relative to GAPDH
and a coincident increased level of p53 message. At day 15, a
developmental stage following a significant amount of organ system
differentiation and morphogenesis(56) , cyclin G1 message again
increased relative to GAPDH while p53 levels remained the same or
slightly decreased. Taken together, cyclin G1 and p53 mRNAs appear to
be independently and differentially regulated during murine embryonic
development.
To determine if the level of cyclin G1 mRNA could be
modulated in cells not expressing the transcriptional activator p53, we
tested the effect of several growth and stimulatory factors on cyclin
G1 expression in the p53 null Bal-17 cells over time. The negative
growth factor TGF- is known to influence the expression of some
cell cycle components (57, 58) and has established
kinetic effects on the cell cycle of murine B
cells(59, 60, 61) . Exponentially growing
cultures of Bal-17 (p53
) and WEHI-231
(p53
) were treated with TGF-
at 1 ng/ml and
aliquots were sampled for cyclin G1 mRNA expression over 24 h.
PhosphorImager analysis of Northern blots of the cyclin G1 relative to
GAPDH (control) mRNA (Fig. 4C) in Bal-17 indicated an
4.6-fold increase relative to untreated cells within 6 h of treatment
followed by a decrease to nondetectable levels at 24 h, a point when
growth inhibition is first noticed (data not shown)(61) .
Similar results were seen in WEHI-231 cells. Although the basal level
of cyclin G1 is higher in WEHI-231, relative to the GAPDH control the
initial level of cyclin G1 mRNA increased 2.5-fold at 6 h
post-treatment followed by a similar decrease thereafter (Fig. 4C). Thus cyclin G1 mRNA expression can be
modulated independent of p53 status in BAL-17 cells.
Figure 5: Nucleotide sequence of human cyclin G2 cDNA and alignment of the predicted G1 and G2 proteins to homologous cyclins. A, the cyclin G2 ORF and immediate 5` and 3` region and the predicted amino acid sequence shown below. The ATG (+1) initiation codon is boxed, in-frame termination codons defining the ORF are shaded, and numbers to the right of the nucleotide sequence are relative to the start codon. The shaded boxed region in the protein sequence denotes the cyclin box-homologous region and the double outlined box in the carboxyl terminus indicates a PEST sequence. The potentially phosphorylated tyrosine residues are indicated by an asterisk above. B, amino acid alignment of rat human and mouse cyclin Gl and G2 proteins to partial human and mouse cyclin A, and Schizosaccharomyces pombe Cig1 and Cig2 proteins based on the crystal structure of human cyclin A (63) . Identical amino acids are shaded and gaps introduced for optimal alignment are indicated by periods. Boxes define putative helical repeats and vertical boxes highlight the constrained alanine residues defining the interhelical crossing points(63) . These are replaced by glutamic or aspartic acid in the C-proximal helices of cyclin G1 and G2. Asterisk (*) indicates conserved residues critical for cyclin A-CDK contact.
Cyclin G1 is a homologue of the fission yeast B-type Cig1(35) . Comparative ``Bestfit'' analysis (GCG) of the peptide sequences in the cyclin box region of cyclin G2 shows that while cyclin G2 has sequence identity with Cig1 (25%), it exhibits higher sequence identity to the fission yeast Cig2 and the budding yeast cyclin Clb-5. Amino acid identity and similarity of cyclin G2 to these yeast cyclins extends beyond the cyclin box region of cyclin G2. Overall identity (and similarity) of cyclin G2 to Cig2 is 25% (and 50%) and to CLb5 is 32% (and 49%), respectively, compared to 19% identity and 41% similarity to Cig1. Previous analysis indicated that Cig1 had considerable sequence identity to cyclin G1 in the carboxyl-terminal region(35) , however, this analysis had not detected a region in the carboxyl terminus of both proteins with the consensus SGXTARQLK(5X)I(6-7X)P which contains a shared potential PKC phosphorylation site.
The same Northern blot membranes
sequentially probed for human cyclin G1 (2.8 kb mRNA), cyclin G2 (2.9
kb), and a control gene, demonstrated obvious differences in their
expression pattern. In contrast to cyclin G1, cyclin G2 was not
prominently expressed in skeletal muscle but was most strongly
expressed in the cerebellum. In addition, cyclin G2 mRNA levels are
high in the spleen, thymus, and prostate relative to cyclin G1,
relative to GAPDH and -actin levels (Fig. 6A, left
panel). Examination of a variety of human B cell lines for
expression of these two cyclins showed striking differences. While
cyclin G1 was expressed at moderate to high levels (relative to GAPDH)
in all of the cell lines examined, cyclin G2 mRNA was primarily
elevated in Daudi cells (Fig. 6A, right panel).
Progressively lower levels of G2 transcript expression were detectable
in Ramos, NALM-6, DG-75, and Raji cells and no significant level of G2
mRNA was observed for IM-9 or T-51 cells. It is important to note that
the Burkitt's lymphoma cell lines Daudi, Ramos, and Raji harbor
either homozygotic or heterozygotic mutant forms of the p53 allele (65, 66, 67) and that the level of p53
expression in these cell lines is independent of their Epstein-Barr
virus status. To address the status of the p53 protein expressed in
these cell lines we performed immunoprecipitations using antibodies
recognizing either mutant or wild-type p53, and examined functional p53
activity by testing for G1 arrest after
-irradiation. Using this
combined analysis the wild-type form of p53 was not detectable in
lysates of the suspected mutants, nor were the p53 mutant cell lines
sensitive to
-irradiation-induced G
arrest (data not
shown). Clearly there is no correlation between the level of either
cyclin G1 or cyclin G2 transcripts and this aspect of p53 function in
these human cell lines.
Figure 6:
Northern blot analysis of human tissues,
cell lines, and cell cycle for cyclin G1, G2, and D2. A,
comparative multiple tissue and multiple B cell line Northern blot
analysis of the distribution and level of cyclin G2 transcript
expression relative to cyclin G1. B, FACS profile of cellular
DNA content from elutriated populations of Jurkat T-cells (left) and human peripheral T-lymphoblasts (right)
used in the corresponding Northern analysis shown below relative to the position of a 2.4-kb marker. Northern analysis of
15 µg of total RNA probed for cyclin G1, cyclin D2, and
-actin. C, line graph comparison of the cyclin G1, cyclin
G2, and cyclin D2 mRNA levels throughout the cell cycle normalized to
the
-actin levels in Jurkat (left) and peripheral
T-lymphoblasts (right).
Northern analysis of cyclin G1 expression in
murine B cells had indicated expression was up-regulated in transition
from early G phase to late G
phase but remained
at constitutively high levels throughout the rest of the cell cycle,
similar to results described for cyclin G1 expression in fibroblasts
following block and release experiments(35, 36) . To
evaluate expression of cyclins G1 and G2 through the cell cycle of
human lymphocytes, Jurkat T-lymphoblastoid and normal human peripheral
T-lymphoblasts were fractionated by elutriation into populations at
progressive phases of the cell cycle. Total RNA was extracted and
examined by sequential Northern blot analysis (Fig. 6B). Cyclin G1 mRNA levels showed moderate
fluctuation during the cell cycle in Jurkat cells, increasing slightly
in early G
(2-fold) to peak levels in late
G
/early S phase and declining to early G
levels
by late G
/M (Fig. 6C). In normal stimulated
T lymphoblasts, cyclin G1 expression was relatively constant following
an initial decline from an early G
phase peak. This pattern
of relatively constant cyclin G1 expression was contrasted by a clear
oscillation in cyclin G2 transcript levels through the cell cycle. In
both Jurkat T cells and normal T-lymphoblasts, cyclin G2 mRNA steadily
increased to peak levels in the mid-S phase and decreased during
G
/M phase progression. Thus cyclin G1 and G2 transcript
were distinguished not only by the distribution and amount in different
tissues and cell types, but also by regulation of their expression
through the cell division cycle.
Cyclins thus far described are characterized by a conserved
cyclin box region of 110 amino acids surrounded by unique
NH
-terminal and COOH-terminal sequences. Within subsets of
cyclins, these flanking sequences also contain either destruction box
or PEST motifs regulating protein stability. Outside of the cyclin box
region, extensive sequence identity (>40%) is not present between
the cyclins of different families, and within families such as the
B-type mitotic cyclins or D-type family of G
cyclins, the
overall amino acid identity is between 45 and 70%. We have described a
new cyclin G1 homologue, cyclin G2, with 60% nucleotide sequence
identity and 53% protein identity (72% similarity) to the cyclin G1.
Cyclin G2 cDNA predicts for a protein of
40 kDa as compared to 34
kDa for cyclin G1, and alignment of the deduced amino acid sequence
from cyclin G2 and G1 ORFs demonstrates significant sequence identity
NH
-terminal as well as COOH-terminal to the cyclin box.
Analogous to the D-type cyclins(2, 21) , the murine,
rat, and human cyclin G1 proteins are nearly identical (>95%), while
human G1 and G2 (and murine G1 and G2 homologues) (
)exhibit
a lower overall identity, suggesting that these two cyclins represent
non-redundant molecules which play discrete functional roles.
Examination and comparison of our sequence for human and mouse cDNAs
encoding cyclin G1 with those previously published (35, 36, 48) has identified a new translation
initiation codon that includes a flanking region
NH-terminal to the cyclin box. This is consistent with the
recently revised coding region for rat cyclin G (37) .
Moreover, analysis of cyclin A-CDK2 structure has demonstrated the
helical region NH
-terminal to the cyclin box is
requisite for cyclin-CDK interaction(63) . Analysis of cyclins
G1 and G2 in relation to the recently resolved crystal structure of
cyclin A (63) , indicates that alanine residues defining the
crossover points in the
2 of the cyclin box for cyclin A are
conserved in Cig1, Cig2, cyclin G1, and cyclin G2, but not among the B
cyclins of higher eukaryotes(5) , while the alanine in
3
helix, defining the cyclin fold, are conserved among all known cyclins
except cyclin F. However, the cyclin fold defining alanines in the
carboxyl proximal
2` and
3` helices of cyclin A are replaced
by glutamic and aspartic acid residues in cyclin G1 and G2, and the
putative inter-helical hinge regions are extended. Based on this
alignment the NH
terminus of cyclin G1 and G2 are spatially
close to and important for the cyclin-CDK interface and the COOH
terminus is spatially removed to the CDK binding site, and may be
available for either substrate or localizing interactions. Cyclins G1
and G2 have the highest sequence identity and similarity to the
mammalian cyclin A proteins and the fission yeast B-type cyclins Cig1
and Cig2, respectively, while cyclin G2 exhibits high sequence identity
to the S-phase cyclin Clb-5 of budding yeast.
Murine and human
cyclin G1 mRNA have transcript sizes 3.4 and 2.8 kb, respectively,
in agreement with the work previously published (36, 48) . Parallel analysis of the human cyclin G2
mRNA defines an
2.9-kb transcript. Our results indicate no obvious
correlation between expression of these cyclins and the proliferative
state of the tissue. Cyclin G1 is expressed at high levels in skeletal
muscle, ovary, kidney, and colon. Cyclin G2, in contrast, is expressed
at high levels in cerebellum, thymus, spleen, and prostate, and at low
levels in skeletal muscle. While it is possible the level of mRNA
detected may reflect a quantity of cycling cells in each tissue,
several of these tissues are primarily composed of terminally
differentiated cells and elevated levels of cyclin G1 in heart muscle,
a tissue lacking self-renewing stem cells(68) , make a
proliferative component unlikely. Cyclin D3 is also increased upon
terminal differentiation(69) . Previous studies using serum
starvation or metabolic blocks to synchronize cells suggested
expression of human and rat cyclin G1 mRNA was either induced in
G
phase to constitutively high levels throughout the cell
cycle, or was unchanged as cells progressed from quiescence through the
proliferative cell cycle. To clarify this we have examined cyclin G1
and G2 expression in the progressive stages of the cell cycle by
partitioning proliferating cells with the less invasive method of
centrifugal elutriation. Under these conditions cyclin G1 mRNA levels
do not change significantly following an apparent induction in early
G1. In contrast, cyclin G2 transcripts do exhibit cell cycle
periodicity, reaching peak levels at mid-S phase. Similar to cyclin G1,
transcripts of the mammalian cyclins C and D1 peak early in transition
to the G
phase of the cell cycle following growth factor
induction and oscillate only minimally throughout the proliferative
cell cycle.
While the deduced amino acid sequence from the newly defined murine, rat, and human cyclin G1 ORFs exhibit neither a canonical destruction box nor PEST sequence, the predicted human cyclin G2 sequence contains a PEST-rich protein destabilizing sequence in its carboxyl terminus. The lack of destabilization motifs in cyclin G1, or any periodicity in its message during the cell cycle, suggests control of its activity may occur at the post-transcriptional or post-translational level. In contrast, both cyclin G2 and the yeast homologue, Cig2, show cell cycle dependent periodicity in their mRNA levels. Moreover, both of these contain protein destabilization motifs, Cig2 a destruction box, and cyclin G2 a PEST-rich sequence, making it likely that the expression of these cyclins is tightly regulated through the cell cycle.
Prior reports (36, 37) identified murine and rat cyclin G1 as a transcriptional target of the p53 tumor suppressor. In agreement with these results, cyclin G1 levels in proliferating murine lymphocytes were paralleled by the presence or absence of wild-type p53 expression. In contrast to high cyclin G1 levels in WEHI-231 cells (determined to express wild-type p53), 6-fold lower levels of cyclin G1 are present in the p53 negative Bal-17 cells. p53 expression in many cell lines has been linked to the induction of cell cycle arrest and apoptosis(38, 70) , and it was proposed that the induction of cyclin G1 by p53 may be part of the pathway leading to arrest and or apoptosis(36) . WEHI-231 cells undergo cell cycle arrest followed by apoptosis upon cross-linking of surface IgM, while Bal-17 cells continue to proliferate upon stimulation of its B cell receptor(52) , thus it is tempting to link wild-type p53 expression and induction of cyclin G1 in WEHI-231 to cell cycle arrest and apoptosis. Establishment of p53 and cyclin G1 expression constructs into Bal-17 should indicate if there is a causal relationship between their expression and programmed cell death in murine B cell development.
Our further analysis shows the relationship between
cyclin G1 transcript levels and expression of wild-type p53 mRNA is
complex and contingent on growth conditions, tissue type, and stage of
embryonic development. During embryonic development, and in tissues
from p53 null mice, cyclin G1 transcript levels are often found inverse
to or unrelated to p53 levels. Elevated levels of cyclin G1 and
suppressed p53 levels are seen at day 7 (gastrulation) of mouse
development. Cell cycle regulation during gastrulation differs from
that at other stages of development (54) and accelerated cell
division is punctuated by contraction of S and G phases
while the typically variable G
phase is relatively
unchanged(55) . Transcript levels of cyclin G1 in tissues from
wild-type, heterozygous, and p53 null mice were reduced in kidney, but
tissues such as brain, stomach, and testis showed no reduction in the
amount of cyclin G1 mRNA upon loss of functional p53, and in some cases
message was increased upon p53 loss. Lack of correlation between cyclin
G1 expression and p53 is also evident in our Northern blot analysis of
cyclin G1 message in Ramos, Daudi, Raji, and Jurkat cells and the
report of abundant cyclin G1 mRNA in SAOS-2 fibroblasts(48) ,
all previously determined to be p53 deficient cell
lines(65, 66, 67, 71) . p53 mutant
alleles may be diverse in their ability to transactivate, yet in two of
the above mentioned cell lines p53 is most likely not acting as the
direct transcriptional activator of cyclin G1. SAOS-2 fibroblasts
completely lack endogenous p53(72) , yet maintain a high level
of cyclin G1 message(48) ; and in Ramos cells the expression of
another direct transcriptional target of p53, the CDK inhibitor
p21
, is not induced by
-irradiation treatment
that up-regulates p53 and p21
in p53 wild-type
cells(70) , indicating that the p53 protein expressed at high
levels in this cell line is not a functional transcriptional activator.
Transcriptional activators, in addition to p53, are likely to be
involved in regulating cyclin G1 expression. The expression of
p21
has been reported to be activated in some cell
lines treated with growth inhibitory
factors(70, 73, 74) , independent of the p53
status of the cell. It has been recently shown that the transactivator
NF-
B binds and activates transcription through the p53 response
element(75) , raising the possibility that cyclin G1
transcription may also be activated by others factors such as
NF-
B.
It is not clear if cyclin G1 expression is linked to
positive or negative growth regulation. Cyclin G1 is induced in both
p53 negative and p53 wild-type murine B cells lines by treatment with
the negative growth factor TGF-. This TGF-
response occurs 18
h prior to any inhibition of cell growth and more than 66 h before
significant G
phase accumulation of TGF-
treated
WEHI-231 cells (61) . It is possible that factors affecting
mRNA stability may also play a role in regulating cyclin G1 expression.
TGF-
related stabilization of mRNAs has been reported and found to
be affected by TGF-
responsive elements present in the 3`-UTR of
these sequences (76, 77, 78) . The 3`-UTR of
several lymphokine and proto-oncogene mRNAs are known to contain
AU-rich elements with the motif AUUUA acting as instability
determinants(49, 50, 51, 79) . The
conserved block of nucleotide sequence containing reiterated copies of
the canonical AUUUA motif present in the 3`-UTRs of human, rat, and
murine cyclin G1 gene transcripts could reflect a region regulating
cyclin G1 mRNA stability.
The function of these cyclins remains
unclear. Although overexpression of cyclin G1 in human osteosarcoma
cells or murine fibroblasts had no observable effect on the cell cycle
progression of these cells(36) , it is possible the
NH-terminal 45 amino acids missing in the cyclin G1
constructs are necessary for structural conformation. Based on
up-regulation by p53, it has been proposed that high levels of cyclin
G1 expression may inhibit the activity of one or more of the
G
-type cyclins by competing for association with a partner
catalytic CDK and thus acting as an anti-cyclin(36) . In this
regard, a possible competitive interaction between cyclin G2, cyclin
G1, and a hypothetical CDK partner could be postulated. Cyclin G2
contains two potentially phosphorylated tyrosine residues suggesting a
role for cyclin G2 in a signal transduction cascade and cyclin G1 may
act as a competitive inhibitor. The presence of the N-X-X-Y
recognition sequence for binding of the PTB domain of the Shc signaling
protein (64, 80) in the carboxyl terminus of cyclin G2
but not cyclin G1 may be biologically relevant.
These cyclins
exhibit differential tissue distribution, cell cycle-regulated
transcript expression, and unique structural features. Our recent
detection of another homologue of cyclins G1 and G2 may expand this
family to at least three members. ()Further experiments to
assess protein abundance, subcellular localization, and associations
with other cell cycle component partners is required to shed further
light on their function.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L49504[GenBank], L49506[GenBank], and L49507[GenBank].