(Received for publication, July 7, 1995; and in revised form, October 19, 1995)
From the
The activation of cyclin-dependent protein kinases (Cdks) is dependent upon site-specific phosphorylation and dephosphorylation reactions, as well as positive and negative regulatory subunits. The human Cdk-activating protein kinase (Cak1) is itself a Cdc2-related cyclin-dependent protein kinase that associates with cyclin H. The present study utilized specific anti-Cak1 antibodies and immunoaffinity chromatography to identify additional Cak1-associated proteins and potential target substrates. Immunoprecipitation of metabolically labeled human osteosarcoma cells revealed a number of Cak1-associated proteins, including p95, p37 (cyclin H), and a 35-kDa protein that was further characterized herein. Microsequence analysis obtained after limited proteolysis revealed peptide fragments that are similar, but not identical to, human and yeast cyclins, thus identifying p35 as a cyclin-like regulatory subunit. The greatest sequence similarity of human p35 is with Mcs2, a yeast cyclin that is essential for cell cycle progression. Immunoaffinity chromatography performed under nondenaturing conditions afforded the isolation of enzymatically active Cak1 from cell lysates, enabling studies of kinase autophosphorylation and comparative substrate utilization. Immunoaffinity-purified Cak1 phosphorylated monomeric Cdc2 and Cdk2, but not Cdk4; the phosphorylation of both Cdc2 and Cdk2 were increased in the presence of recombinant cyclin A. These studies indicate that the Cak1 catalytic subunit, like Cdc2 and Cdk2, associates with multiple regulatory partners and suggests that subunit composition may be an important determinant of this multifunctional enzyme.
Critical transitions in the eukaryotic cell cycle are regulated
by patterns of protein phosphorylation events governed predominantly by
the catalytic activities of cyclin-dependent protein kinases (Cdks) ()(Norbury and Nurse, 1992; Meyerson et al., 1992;
Reed, 1992; Pines, 1993; Dorée and Galas, 1994).
The activities of these Cdks are themselves regulated by site-specific
phosphorylation-dephosphorylation reactions (Krek and Nigg, 1991;
Norbury et al., 1991; Solomon, 1993) as well as the
association with positive (i.e. cyclins) (Hunt, 1991; Wu et al., 1993; Sherr, 1993; Draetta, 1994) and negative (i.e. Cdk inhibitors) (Elledge and Harper, 1994; Peter and
Herskowitz, 1994; Xiong et al., 1993) regulatory subunits. An
essential phosphoregulatory site, deduced from genetic studies in
yeast, was identified within the T-fold of kinase subdomain VIII
(Marcote et al., 1993; DeBondt et al., 1993) of Cdc2
(at Thr-161) and Cdk2 (at Thr-160) and whose phosphorylation is
required for the generation of Cdc2/Cdk2 kinase activity (Krek et
al., 1992; Gu et al., 1992; Connell-Crowley et
al., 1993). Subsequent studies have identified a distinctive
Cdc2/Cdk2-activating protein kinase (CAK) in Xenopus oocytes,
MO15 (Shuttleworth et al., 1990; Poon et al., 1993;
Solomon et al., 1993), and human tumor cells (Solomon et
al., 1993; Williams et al., 1993a, 1993b). The catalytic
subunit of the human CAK (Cak1) has been cloned and is found to be
structurally related to Cdc2(Hs) (Wu et al., 1994). Human Cak1
has been shown to complex with at least one regulatory subunit, cyclin
H (Fisher and Morgan, 1994; Mäkelä et al., 1994), revealing a vectorial cascade of
cyclin-dependent protein kinases.
Other recent studies suggest that Cak1 functions in transcriptional regulation, DNA repair, and tumor suppression, in addition to its role in regulating cell cycle progression.
However, numerous questions remain regarding the subunit configuration(s), biochemical activation, substrate specificity, and physiological targets of this key regulatory enzyme. Pronounced disparities in the published literature concerning the molecular mass of the Cak1 complexes (Solomon et al., 1992; Poon et al., 1993; Williams et al., 1994; Wu et al., 1994), the nature and identity of its regulatory subunit(s) (Mäkeläet al., 1994; Tassan et al., 1994), the phosphorylation of putative target substrates, including monomeric Cdc2 (Dorée and Galas, 1994; Fisher and Morgan, 1994) and Cdk4 (Kato et al., 1994; Matsuoka et al., 1994), and the regulation of its enzymatic activity (Fisher and Morgan, 1994; Matsuoka et al., 1994; Williams et al., 1994), suggest that our understanding of this key regulatory enzyme system is still incomplete.
Using the
deduced amino acid structure of (Hs)Cak1 (Wu et al., 1994), we
developed anti-peptide antibodies that are selective for the
p42 subunit and have characterized the
performance of these antibodies for both immunoprecipitation and
Western blotting. In this study, we used anti-Cak1 antibodies and
immunoaffinity chromatography to investigate the enzyme activities of
purified Cak1 and to further characterize Cak1-associated proteins. In
addition to verifying high molecular weight complexes reported
previously (Williams et al., 1993a, 1993b) and the association
of a 37-kDa protein identified as cyclin H
(Mäkeläet al., 1994;
Fisher and Morgan, 1994), we detected a distinctive 35-kDa protein in
association with p42
. Isolation, fragmentation,
and microsequence analysis of p35 revealed that this Cak1-associated
protein exhibits amino acid sequence homologies to human cyclin H and
yeast Mcs2; indicating that p35 is indeed a cyclin-like
regulatory subunit that is related to, but distinct from, cyclin H.
For
immunoprecipitations, MG-63 and TE-85 cells were cultured and lysed as
described above, and the lysate was clarified by incubation with 50
µl (1:1 slurry in (1) PBS) of protein A-Sepharose CL-4B on
ice for 15 min. Immunoprecipitations and affinity precipitations were
performed by incubating aliquots of the precleared lysate (supernatant)
with either no antibodies (control), specific antibodies, or specific
peptide-blocked antibodies for 20 min in ice with periodic mixing. The
resulting immune complexes were collected by the addition of 50 µl
(1:1 slurry in (1
) PBS) of protein A-Sepharose CL-4B, incubation
in ice for 20 min, followed by centrifugation. After washing each
pellet 4 times with 750 µl of ice-cold Fast Q Buffer (20 mM Tris-HCl, pH 7.6, 1 mM dithiothreitol, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
leupeptin, 0.1 mM sodium orthovanadate) containing 50 mM NaCl, and once in 750 µl of ice-cold kinase assay buffer, each
pellet was resuspended in 50 µl of (1
) SDS sample buffer
(62.5 mM Tris-HCl, pH 6.8, 40 mM dithiothreitol, 2%
SDS, 0.025% bromphenol blue, 10% glycerol) containing 10 mMN-ethylmaleimide (to minimize dissociation of IgG chains),
heat-denatured for 2 min at 95 °C, and the solubilized proteins
were analyzed by SDS-PAGE and Western blotting, as described by
Williams et al.(1992). The specific immunoreactivity of the
newly developed anti-Cak1 antibodies were validated by competition of
the antibody with the immunizing peptide as follows: approximately 400
µg of the immunizing peptide was mixed with anti-Cak1 IgG and
preincubated for 30 min at 30 °C prior to separate parallel
incubations of both blocked and nonblocked antibodies for 30 min on
Western blots. The resulting Western blots were developed under
identical conditions.
Hela S-3 cells (5-7 g pellets)
were stored at -70 °C until use. The cells were lysed in 20
volumes of IP buffer at 4 °C and cleared by centrifugation (10 min
20,000
g) and filtered through 0.45 µm
prior to immunoaffinity column chromatography. After the entire
clarified cell lysate has passed through the column, the column was
developed as follows: a 10-volume wash with IP buffer followed by 5
volumes of 1
PBS, followed by 10 volumes of 0.3 M NaCl
in IP buffer, followed by 10 volumes of 0.3 M NaCl, 30%
ethylene glycol in IP buffer, followed by 5 volumes of 1
PBS,
followed by 10 volumes of 1 M MgCl
in IP buffer,
followed by 5 volumes of 1
PBS, followed by elution with 0.1 M glycine, pH 2.7 (collected in 0.5-ml fractions). The
glycine-eluted fractions containing proteins were pooled and
concentrated in Amicon microconcentrators, and samples of each wash
were analyzed by Western blotting. Enzymatically active Cak1 was
recovered in both the NaCl/ethylene glycol and the MgCl
eluates, as well as the NaCl wash, indicating that a substantial
proportion of the anti-Cak1 peptide antibodies were low to moderate in
their affinity (Kellogg and Alberts, 1992) and enabling the
purification of Cak1 under nondenaturing conditions.
Figure 1:
Design and
application of specific anti-Cak1 antibodies. A, alignment of
the C-terminal domains adjacent to the conserved subdomain XI of
identified human Cdks. The primary structure of the synthetic peptide
used to raise specific anti-Cak1 antibodies is underlined. Double underlined is the putative PKA phosphorylation site
located within the bipartite nuclear localization motif. B,
titration of Cak1 detection upon Western blotting by polyclonal
anti-Cak1 antibodies raised against the immunizing peptide. Note
comparative cross-reactivity with Xenopus M015, which exhibits
a similar but nonidentical C terminus (see Shuttleworth et
al., 1990). C, Western blotting of cell lysates obtained
from a variety of normal and neoplastic cell lines with
affinity-purified anti-Cak1 antibodies. Each lane was loaded
with 10 µg of protein of the respective detergent lysate: normal
rat kidney fibroblast (NRK-49F), rat osteosarcoma (UMR-106), normal human diploid fibroblast (WI-38),
human osteosarcoma (MG-63, SAOS-2, TE-85, U2OS), Ewings
sarcoma (EW-1), mammary carcinoma (MDA-MB),
epithelioid carcinoma (A-431), pancreatic carcinoma (HS-766T). While the anti-Cak1 antibodies detected a protein
of the expected size (p42) in all samples,
additional bands of immunoreactivity migrating at
36 and
55
kDa were pronounced in the majority of the human cancer-derived cell
lines (see lanes 5-11). Also note the additional band at
43 kDa that appears to be unique to the rat osteosarcoma cell line (lane 2).
The anti-Cak1 antibodies efficiently precipitated
the native protein, enabling further studies of Cak1-associated
proteins. Immunoprecipitation experiments of
[S]Met/Cys-labeled osteosarcoma cells revealed
two prominent proteins, at 37 and 35 kDa, respectively, that associate
with Cak1 in potentially stoichiometric amounts (Fig. 2A), and the former has been identified as cyclin
H (Mäkeläet al.,
1994; Fisher and Morgan, 1994). A heavily labeled protein of
80-90 kDa, as well as several proteins in the 50-55-kDa
range, were also identified as potential Cak-associated proteins. The
specificity of the immunoprecipitation assay was confirmed by the use
of affinity-purified antibodies (Fig. 2A, lane
3) and peptide-blocked antibodies (Fig. 2A lane 4), as well
as minus antibody controls. Western blotting of the immunoprecipitates
confirmed that p42
was the only immunoreactive protein
recognized by these antibodies (Fig. 2A, lanes
5-8) further indicating that the profile of proteins
represented are physically associated with Cak1 (see
``Discussion''). Further studies of Cak1-associated proteins
in highly synchronized MG-63 osteosarcoma cells (Fig. 2B) confirmed the constitutive expression of
p42
(Wu et al., 1994) and demonstrated that
both p37 (i.e. Cyclin H) and p35 remain constant in their
association with Cak1 throughout the cell cycle. However, the relative
abundance of the 90-95-kDa protein, as well as its
electrophoretic migration pattern, appears to shift as cells proceed
from G
to S phase.
Figure 2:
Identification of Cak1-associated proteins
in human osteosarcoma cells. A, asynchronous cultures of TE-85
osteosarcoma cells were metabolically labeled with
[S]Met/Cys, and the washed cells were lysed and
immunoprecipitated with anti-Cak1 antibodies (IgGs, lanes 2 and 6) or affinity-purified anti-Cak1 antibodies (Affi, lanes 3 and 7). The specificity of
the immunoprecipitation was confirmed by minus antibody (Control, lanes 1 and 5) and by
precipitation with peptide-blocked primary antibodies (+Peptide, lanes 4 and 8). The
resulting immune complexes were analyzed by autoradiography (left
panel). Of particular interest are the three prominent bands
migrating at
35, 37, and 42 kDa, respectively, as well as several
proteins of higher molecular mass. Western analysis of the resulting
immune complexes (right panel) identifies the 42-kDa protein
as Cak1 and further indicates that p35 and p37 are Cak1-associated
proteins. B, cell cycle dependence was examined in highly
synchronized MG-63 cells (Carbonaro-Hall et al., 1993), which
were lysed and analyzed by immunoprecipitation with (+) and
without(-) anti-Cak1 antibodies. Based on SDS-PAGE and
autoradiography (left panel), Cak1 (p42) appears to be
constitutive in the cell cycle, as are its associated proteins, p37 (i.e. cyclin H), p35, p55, and p90, although p90 undergoes
appreciable shifts in electrophoretic mobility in the transition from
G
to S phase. Western analysis (right panel)
identifies the Cak1-associated p37 as cyclin
H.
Figure 3:
Immunoaffinity chromatography isolates
Cak1 complexes from Hela cell lysates. Approximately 7 g (wet weight)
of frozen Hela cells were lysed, filtered, and applied to an anti-Cak1
affinity matrix, as described under ``Experimental
Procedures.'' The affinity-purified protein complexes were eluted
with 100 mM glycine, pH 2.7 (lane 4), followed by
Western analysis of the various fractions and eluates for p42/Cak1
immunoreacted with the anti-Cak1 antibodies. The efficiency of Cak1 in
depleting p42/Cak1 from the crude lysates is shown (compare lanes 1 and 2). Protein staining of part of the eluate indicate
the relative abundance of the three proteins bands of particular
interest: p42, identified as Cak1; p37, identified as Cyclin H; and
p35, which remains to be characterized. The band of protein at 35
kDa was excised and subjected to limited proteolysis by endoprotease K,
followed by HPLC separation of the digests and microsequencing of the
resulting peptides, as described under ``Experimental
Procedures.'' Five peptide sequences were obtained, as shown. (Parentheses indicate
uncertainty.)
Figure 4: Amino acid sequence alignment of peptides derived from the Cak1-associated 35-kDa protein with identified cyclins. The amino acids sequences of three of the five peptides obtained are aligned with three of their nearest cyclin relatives: the yeast Mcs2 (Molz and Beach, 1993), the human cyclin H (Fisher and Morgan, 1994), and human cyclin C (Fisher and Morgan, 1994). The homology to Mcs2 is greater than the homology to human cyclin H and human cyclin C, suggesting that p35 may be a human homologue of the yeast cyclin, Mcs2.
Figure 5:
Immunoaffinity chromatography yields
active Cak1 complexes from Hela cell lysates. A, approximately
7 g (wet weight) of frozen Hela cells were lysed, filtered, and applied
to an anti-Cak1 affinity matrix, as described under ``Experimental
Procedures''; the column was washed with 300 mM NaCl (lane 3), and the affinity-purified protein kinase complexes
were eluted sequentially with 300 mM NaCl plus 30% ethylene
glycol (lane 4), 1 M MgCl (lane
5), and 100 mM glycine, pH 2.7 (lane 6),
followed by Western analysis of the eluates for
p42
. The efficiency of Cak1 depletion from the
crude lysates (compare lanes 1 and 2), taken together
with the elution of the bulk of the bound enzyme under nondenaturing
conditions, indicates that the anti-Cak1 antibodies were uniformly low
to moderate in their affinity. B, immunoaffinity-purified Cak1
(MgCl
fractions) were utilized as a source of enzyme for in vitro kinase assays performed in the presence (+) or
absence(-) of recombinant cyclin A with either glutathione S-transferase-Cdc2, glutathione S-transferase-Cdk2,
or an Ala-160 mutant of glutathione S-transferase-Cdk2 as
substrates for Cak1. The migration of the respective recombinant
proteins upon SDS-PAGE was visualized by Coomassie Blue stain (arrows, left panel), while the kinase reaction was
evaluated by autoradiography (right panel). Under these
conditions, both Cdc2 and Cdk2 are more readily phosphorylated by Cak1
in the presence of cyclin A (compare lanes 1 and 2 and lanes 3 and 4), while the Cdk2-A mutant was
enzymatically active toward cyclin A (see lane 5) without
Thr-160 phosphorylation (Connell-Crowley et al., 1993). Note
discernable autophosphorylation of immunopurified
p42
. C, comparative utilization of
recombinant Cdc2, Cdk2, and Cdk4 as Cak1 substrates was examined by
incubation of the respective recombinant Cdk substrates in the presence
(+) or absence(-) of immunoaffinity-purified Cak1 followed
by SDS-PAGE and autoradiography. Under these conditions, monomeric Cdc2
and Cdk2, but not Cdk4 was readily phosphorylated by Cak1
complexes.
The immunoaffinity-purified Cak1 preparations exhibited
phosphorylation of the p42 monomer in in vitro kinase assays (Fig. 5B), resulting presumably from
autophosphorylation, although heterologous phosphorylation by a tightly
associated Cak1 kinase cannot be ruled out. Furthermore, the
immunopurified Cak1 preparations were active in phosphorylating Cdc2
and Cdk2, but not Cdk2/160-Ala, even in the presence of recombinant
cyclin A (Connell-Crowley et al., 1993). Phosphorylation of
both Cdc2 and Cdk2 was markedly increased by the addition of
recombinant Cyclin A to the reaction mixtures. In agreement with recent
reports using purified materials (Dorée and
Galas, 1994), but in contrast to those using reconstituted
Cak1
cyclin H complexes (Fisher and Morgan, 1994), these
immunopurified Cak1 preparations phosphorylated both monomeric Cdc2 and
Cdk2 but not Cdk4 (see Fig. 5C), suggesting that they
may include regulatory and/or targeting subunits that are missing from
the reconstituted Cak1
cyclin H complexes.
Subunit configuration and site-specific phosphorylation
reactions regulate the enzymatic activities of the cyclin-dependent
protein kinases p34 and p33
. Our previous
studies of Cdc2 phosphorylation led to the isolation (Williams et
al., 1993a, 1993b) and molecular cloning (Wu et al.,
1994) of the human Cdk-activating kinase, p42
, a
homologue of Xenopus MO15 (Shuttleworth et al., 1990)
that targets the phosphoregulatory sites within subdomain VIII of Cdc2
and Cdk2 (Solomon, 1993, 1994; Solomon et al., 1993; Poon et al., 1993). The striking homology of (Hs)Cak1 to (Hs)Cdc2
(Wu et al., 1994), together with the identification of Cyclin
H as a prospective regulatory partner (Fisher and Morgan, 1994;
Mäkeläet al., 1994),
support the concept that Cdks participate in a vectorial kinase
cascade.
Recent studies have indicated that Cak1MO15, by
association with the TFIIH transcription factor, may also function as
the RNA polymerase II C-terminal domain kinase (Roy et al.,
1994; Feaver et al., 1994), linking Cak1 activity to the
control of transcription, as well as cell cycle progression. Taken
together with the seminal observation that M015 mRNA is, by virtue of
alternative start sites (Shuttleworth et al., 1990), capable
of yielding two different forms of the kinase, these findings raise the
possibility that structurally and functionally distinct isoforms of
Cak1 may participate in transcriptional regulation vis à vis cell cycle control pathways. Alternatively,
the association of Cak1 with multiple regulatory subunits (i.e. cyclin-like proteins) may provide additional regulatory and or
targeting features, as has been demonstrated for Cdk2 (see Peeper et al. (1993)).
The utility of the antipeptide antibodies in immunoprecipitation studies was further exploited in immunoaffinity chromatography. The ability of these antipeptide antibodies to release enzymatically active Cak1 complexes under nondenaturing conditions indicate that the bulk of the precipitable antibodies within the IgG fractions were low to moderate in affinity (Kellogg and Alberts, 1992). Immunoaffinity-purified Cak1 complexes were capable of phosphorylating monomeric Cdc2 and Cdk2, but not Cdk4 (see Fig. 5C), which is consistent with observations based on purified materials (Williams et al., 1994; Dorée and Galas, 1994) but is strikingly different from the substrate specificity of reconstituted Cak1 (Fisher and Morgan, 1994), indicating additional regulatory features that remain to be identified.
These studies, taken together with other recent findings, support the concept that the Cdk-activating kinase system is highly regulated and multifunctional. The suggestion that Cak1 may be a primary RNA polymerase II C-terminal domain Kinase (Feaver et al., 1994; Roy et al., 1994) points to a major role in transcriptional regulation. CAK activity is associated with the transcription initiation factor TFIIH, which also functions in nucleotide excision repair (Hanawalt, 1994). Thus, in addition to its classic role as the upstream activator for Cdks, and hence crucial to cell cycle progression, Cak1 appears to have a function in DNA transcription, fidelity, and repair mechanisms. Thus, the demonstration that the Cak1 catalytic subunit forms stable complexes with multiple cyclin-like regulatory partners may provide a mechanistic basis for substrate targeting and/or specificity (see Peeper et al., 1993) that would be predicted for a multifunctional enzyme system.