HCF-1 is abundant in rapidly cycling cell types, such as fetal tissue and immortalized cell lines, and a single mutation in the only copy of HCF-1 in one of these lines, BHK, causes the cells to arrest in a G0-like state. To investigate the state of HCF-1 in wild-type G0 cells, PBMCs from whole blood were cultured in the presence and absence of the mitogen PHA. Whole-cell extracts were made from the cultures at a series of time points following mitogenic stimulation and probed by Western blot for the presence of HCF-1 (Fig. A and B). Extracts from unstimulated PBMCs contained only a 50-kDa protein that cross-reacted with HCF-1 antiserum 2159 (HCFp50) (Fig. A, lane 1). Upon mitogenic stimulation, HCFp50 disappeared from extracts within 24 h, and the characteristic higher-molecular-weight forms of HCF-1 appeared (Fig. A, lanes 2 to 6). HCFp50 is derived from the N terminus of HCF-1, as it is detected by antiserum 2159, which was raised against amino acids 384 to 1015 of the N terminus, and not by αrHCF, antiserum prepared against the C terminus of HCF-1 (Fig. B, lane 1). The typical set of HCF-1 C-terminal polypeptides were present in extracts by 24 h after mitogenic stimulation of PBMCs, along with the higher-molecular-weight N-terminal polypeptides (Fig. A and B, lanes 2 to 6). HCFp50 apparently constitutes a unique form of HCF-1 that is only present in whole-cell extracts from unstimulated PBMCs which are in G0.
FIG. 1 Presence of a unique 50-kDa fragment of HCF-1, HCFp50, in unstimulated PBMC extracts. (A and B) PBMCs were mitogen stimulated for the times shown, and whole-cell extracts were prepared, separated by SDS-PAGE, and blotted with HCF-1 antiserum 2159 (A) (more ...)
Interestingly, a Western blot of unstimulated PBMC cells which were isolated and boiled immediately after donation in SDS loading buffer revealed that full-length N-terminal and C-terminal fragments of HCF-1 were present in the cells, along with HCFp50 (Fig. C, lanes 1 and 4). In the previous experiment, apparently either these high-molecular-weight proteins were not extracted from the cells, or the polypeptides were extracted and then converted to HCFp50 in the extract. When cycling HeLa and 293 cells were boiled directly in SDS loading buffer, only high-molecular-weight HCF-1 N- and C-terminal polypeptides were detected (Fig. C, lanes 2 and 3 and lanes 5 and 6, respectively). When unstimulated PBMCs were separated into nuclear and cytoplasmic fractions, HCFp50 was detected primarily in the cytoplasmic fraction (Fig. D, lane 2), while the larger N-terminal polypeptides were present exclusively in the nuclear fraction (lane 1). The majority of C-terminal polypeptides were retained in the nucleus, although a small portion were extracted into the cytoplasmic fraction (Fig. D, lanes 3 and 4). The detection of some C-terminal polypeptides in the cytoplasm (which almost certainly results from limitations in the fractionation protocol), but none of the large N-terminal polypeptides, most likely reflects a difference in the sensitivities of the two antibodies used for the Western blots. The important conclusion arises from the finding that a large majority of the HCFp50 fractionates in the cytoplasm, suggesting that HCFp50 is in a different subcellular location or state than the full-length HCF-1 polypeptides.
We were interested in determining whether the 50-kDa fragment of HCF-1 observed in unstimulated PBMCs could have arisen via proteolysis of the larger HCF-1 polypeptides. A mixing experiment was performed in which PBMC whole-cell extract that had been mitogen stimulated for 120 h (T120), and thus contained the larger HCF-1 polypeptides, was mixed with an equal volume of unstimulated PBMC extract (T0), which contained only HCFp50. After incubation for 1 h at 37°C, the larger HCF-1 N-terminal polypeptides from the T120 extract had been degraded, as they were undetectable by Western blot with antiserum 2159 (Fig. A, lanes 1 to 3). Similarly, the HCF-1 C-terminal polypeptides, which were present in the T120 extract but not in the T0 extract, were also degraded upon incubation with the T0 extract (Fig. B, lanes 1 to 3). Whether the 50-kDa fragment detected by antiserum 2159 was produced during degradation of the larger HCF-1 N-terminal polypeptides could not be determined due to the presence of HCFp50 in the T0 PBMC extract. To address this question, an HA-tagged 110-kDa N-terminal fragment of HCF-1 (HA-HCF1–1019)was synthesized in the presence of 35S-labeled methionine, using in vitro translation. A small amount of the lysate containing HA-HCF1–1019 was incubated with either the PBMC G0 extract or the PBMC mitogen-stimulated cycling extract (Fig. C). Each mixture was immunoprecipitated with an anti-HA antibody and resolved by SDS-PAGE. A 50-kDa band was immunoprecipitated following incubation with the G0 PBMC extract (Fig. C, lane 2). The full-length radiolabeled N terminus was immunoprecipitated following incubation with a PBMC mitogen-stimulated cycling extract (Fig. C, lane 1). Because the 50-kDa fragment retained the HA tag from the in vitro-translated HA-HCF1–1019, it was derived from the N terminus of the protein. These results indicate that G0 PBMC extracts contain a proteolytic activity that can generate HCFp50 from larger N-terminal polypeptides.
FIG. 2 HCFp50 can be formed by proteolysis. (A and B) Western blots with antisera 2159 (A) and αrHCF (B) of PBMC whole-cell extracts which were mitogen stimulated for 0 h (lane 1) or 120 h (lane 2) or with a mixture of the two (lane 3). (C) In vitro-translated (more ...)
The high-affinity site of interaction between HCF-1 and VP16 has been mapped to the N-terminal kelch domain, which HCFp50 should encompass. To confirm that HCFp50 is capable of binding VP16, a GST pull-down experiment was performed (Fig. A). PBMC G0 extract was incubated in the presence or absence of GST-VP16, glutathione-Sepharose was added to the reaction mixture, and the fraction bound to the beads was separated by SDS-PAGE. A Western blot performed with antiserum 2159 detected a single 50-kDa species (Fig. A, lane 2). When GST-VP16 was left out of the mixture, no proteins were detected with the 2159 antiserum (Fig. A, lane 1). This confirms the anticipated interaction between HCFp50 and VP16.
FIG. 3 Association of HCFp50 with VP16, and formation of C1 complex with VP16, Oct-1, and DNA. (A) Pull-down by glutathione-Sepharose of HCFp50 from unstimulated PBMC extracts in the absence (lane 1) and presence (lane 2) of GST-VP16. (B) PBMC extracts that (more ...)
contains the kelch repeats of HCF-1 and is able to bind VP16, it should also be active for C1 complex formation. To test if this was the case, extracts of unstimulated PBMCs from each time point following mitogen stimulation were assayed by EMSA in the presence of VP16, and a radiolabeled oligonucleotide containing the α/IE promoter element (Fig. B). A faster-migrating form of the C1 complex (designated C1*) was observed when the binding reaction mixture contained extract from unstimulated PBMCs (Fig. B, lane 3). The C1 complexes formed with extracts from each successive time point were of a slower mobility than the one immediately preceding it (Fig. B, lanes 5, 7, 9, 11, and 13), approaching the mobility of the C1 complex formed from a HeLa extract (lane 15). Both the C1* complex and the other C1 complexes were VP16 dependent (even-numbered lanes) and could be supershifted with 2159 antibody (data not shown), indicating the presence of HCF-1 N-terminal polypeptides, as would be expected in the VP16-dependent complexes. Interestingly, the difference in mobility of the C1* complexes formed from extracts of mitogen-stimulated PBMCs at different time points did not correlate simply with the size of the HCF-1 detected in these extracts (see Fig. A and B). For instance, the T24
extract contained N- and C-terminal fragments of HCF-1 that appeared to be full-length, but the C1 complex formed from this extract was of a faster mobility than the C1 complex from HeLa cells. The mobilities of the C1 complexes formed from mitogen-stimulated PBMC extracts decreased at successive time points, as the level of full-length HCF-1 increased. HCF-1 purifies from HeLa cells as a large complex, probably consisting of oligomers of the protein. In addition, reconstitution of the C1 complex with material prepared from insect cells suggests that other proteins in addition to HCF-1, Oct-1, and VP16 may associate with the core C1 complex (8
Finally, we investigated whether the generation of HCFp50 is unique to PBMCs. The human secondary cell line WI38 was cultured for 14 days in either high serum, in which they will continue to cycle, or low serum, which causes them to arrest in G0. FACS analysis showed that the WI38 cells grown in high serum contained 23% S-phase cells, while those grown in low serum contained only 2% S-phase cells, indicating that the WI38 cells grown in low serum were in fact arrested (data not shown). Extracts from these cells were analyzed for HCF-1 by SDS-PAGE. Probing of a Western blot with antiserum 2159 revealed that the serum-starved WI38 cells also contain a 50-kDa species, similar to the fragment found in unstimulated PBMCs (Fig. A, lane 2). The serum-starved WI38 extracts also contain very low levels of C-terminal polypeptides from HCF-1, also similar to unstimulated PBMCs (data not shown). When nuclear and cytoplasmic extracts from the serum-starved WI38 cells were analyzed, the 50-kDa species was found in the cytoplasm (Fig. B, lane 2), while the larger HCF-1 N-terminal polypeptides were predominantly nuclear (data not shown). These data suggest that G0 cells contain a unique 50-kDa form of HCF-1, HCFp50, which is derived from the N terminus of the protein, and that this protein appears to be present in the cytoplasm of these cells, in contrast to the nuclear localization of the larger HCF-1 N-terminal polypeptides.
FIG. 4 Appearance of HCFp50 in serum-starved WI38 cells. (A) Western blot of WI38 cells grown in 10% (lane 1) or 0.1% (lane 2) serum with antiserum 2159. (B) Western blot of nuclear (lane 1) and cytoplasmic (lane 2) fractions of WI38 cells grown (more ...)
It has previously been reported that deletion of the nuclear localization signal of HCF-1 causes the protein to localize to the cytoplasm. Expression of this form of HCF-1 also sequestered coexpressed VP16 in the cytoplasm (11
). To test whether HCFp50
would have a similar activity, COS-1 cells were transfected with HA-tagged HCF-1 or with HA-tagged HCF1–460
and stained with monoclonal antibody 12CA5 (Fig. A). This N-terminal fragment of HCF-1 was chosen because of its close approximation to the size of HCFp50
(as determined by SDS-PAGE) and corresponding location within HCF-1. Cells transfected with a plasmid encoding HA-HCF-1 and immunostained with monoclonal antibody 12CA5, which recognizes the HA tag, showed an almost exclusively nuclear localization, with occasional staining of both the nucleus and cytoplasm (Fig. A, left panel). In contrast, cells transfected with a plasmid encoding HA-HCF1–460
showed staining in the cytoplasm, with little or no staining in the nucleus (Fig. A, right panel). When a plasmid encoding VP16 was cotransfected with full-length HA-HCF-1 (Fig. B), VP16 protein accumulated in the nucleus (middle panel) along with HA-HCF-1 (left panel; DAPI staining shown in the right panel). In contrast, when a plasmid encoding VP16 was cotransfected with a plasmid encoding HA-HCF1–460
(Fig. C), VP16 was distributed between the cytoplasm and nucleus (middle panels, top and bottom). The cytoplasmic VP16 was accumulated in a pocket of very bright staining which was coincident with a very strong HA-HCF1–460
signal (left panels, top and bottom; DAPI staining shown in the right panels), suggesting that the two proteins interact in the cytoplasm. The VP16 that is visible in the nucleus is likely to have been imported by the endogenous HCF-1 present in these cells.
FIG. 5 (A) Immunofluorescence of transfected HA-HCF-1 (left panel) and HA-HCF1–460 (right panel) in COS-1 cells with monoclonal antibody 12CA5. (B) Immunofluorescence of HA-HCF-1 cotransfected with VP16 in COS-1 cells; HA-HCF-1 was detected with antibody (more ...)