The MRP-8/14 complex has been shown to inhibit CKII (38
). Since we have shown that the MRP-8/14 complex is expressed in normal epithelial cells that may be a target for HPV infection, we investigated the role of the MRP-8/14 protein complex in CKII inactivation and HPV16 E7 phosphorylation. The results presented here demonstrate that the MRP-8/14 protein complex inhibits HPV16 E7 phosphorylation in vivo and in vitro. MRP-8/14 inhibited CKII activity, which in turn was correlated with inhibition of E7 phosphorylation. MRP-14 alone also showed an inhibitory effect on CKII activity and E7 phosphorylation in vitro. However, MRP-14 did not show a significant inhibitory effect on CKII activity in vivo, possibly due to inefficient uptake. The exact mechanism by which MRP-14 or MRP-8/14 inactivates CKII is not yet clear. Murao et al. showed that MRP-8/14 inhibits only CKI/II but not other enzymes, including cyclic AMP-dependent protein kinase, PKC, v-abl tyrosine kinase, or insulin receptor protein kinase (38
). Our data also confirmed that MRP-8/14 inhibited CKII but not PKC or CaM kinase II, indicating that MRP-8/14's inhibitory effect on CKII was highly specific.
Massimi and Banks (32
) showed that CKII differentially phosphorylates E7 during the cell cycle; the highest level of E7 phosphorylation occurs during the G1
phase at position Ser31/32 by CKII, and once cells enter S phase E7 phosphorylation decreases dramatically. However, E7 is phosphorylated again in S phase by an unknown kinase at position Ser71, and the functional significance of this phosphorylation is not understood. Analysis of E7 phosphorylation during the cell cycle in the presence of exogenous MRP-8/14 showed that MRP-8/14-mediated inhibition of E7 phosphorylation occurs at the G1
phase of cell cycle. Activation of endogenous as well as uptake of exogenous MRP-8/14 did not inhibit E7 phosphorylation in S phase, which occurs through a protein kinase other than CKII. This may explain why we observed only 40 to 50% inhibition of the steady-state level of E7 phosphorylation in HSC-3 cells exposed to MRP-8/14. MRP-8/14-mediated hypophosphorylation of HPV16 E7 during transition from G1
into S phase may play an important role in reducing E7's activation of cell cycle progression.
It is possible that MRP-8/14-mediated CKII inactivation and E7 hypophosphorylation in vivo may be modulated by other cellular proteins that interact with CKII and/or E7. For example, the S100A4 protein mst1 interacts with the regulatory β-subunit of CKII and inhibits CKII-mediated phosphorylation of nonmuscle myosin heavy chain (26
). The p21WAF1/CIP1
inhibitor of cyclin-dependent kinases also interacts with the β-subunit of CKII (19
) and down-regulates the kinase activity of its α-subunit, and thereby it inhibits CKII-mediated phosphorylation of casein and p53 (19
). Therefore, the potential intracellular interaction of MRP-8/14 with CKII may occur in the presence of other proteins, such as mst1 and p21WAF1/CIP1
, and hence its anti-E7 phosphorylation function may be regulated by those and perhaps by other proteins that interact with CKII and E7.
Absence of MRP expression was correlated with activation of kinase activity of CKII. In HPV-infected MRP-negative immortalized cell lines, CKII activity was four- to fivefold higher than in normal epithelial cells that expressed MRP-8 and MRP-14 proteins. Treatment of HPV-infected MRP-negative cells with exogenous MRP-8/14 complex led to inhibition of CKII activity in HPV16- and HPV18-infected cervical epithelial cells. Activation of endogenous MRP-8/14 also inhibited CKII activity and HPV16 E7 phosphorylation in HPV-negative HSC-3 oral squamous epithelial tumor cells. These data indicate that MRP-8/14 may play a role as a strong intracellular factor that may negatively regulate the activity of CKII.
It has been shown that treatment of various normal and transformed cell lines with 50 to 200 μg of MRP-8/14 per ml within 18 to 48 h leads to inhibition of DNA synthesis and cell growth (62
). The minimum effective concentration to inhibit cell growth was approximately 50 μg/ml. We have shown here that prolonged uptake of a much lower concentration (1 to 10 μg/ml) of MRP-8/14 for 7 to 14 days by HPV-positive and HPV-negative tumor cells, as well as normal epithelial cells, caused detachment of cells from the substratum and inhibited their growth. CKII is a highly pleiotropic enzyme that phosphorylates more than 160 cellular proteins with a wide variety of functions, with consequent effects on gene expression, protein synthesis, cell cycling, and differentiation (15
). Therefore, the antiproliferative effect of MRP-8/14 during prolonged treatment may be due to its inactivation of CKII, which is required for cell proliferation and cell viability in both normal and cancer cells. However, MRP-8/14 more strongly inhibited growth of HPV-positive cells than HPV-negative cells, a difference that may have been due to their effect on E7. Our data therefore suggest that MRP-8/14-mediated CKII inactivation may lead to two groups of downstream antiproliferative effects. The first group of effects is more general, reflecting inhibition of phosphorylation of CKII substrates, i.e., housekeeping proteins that are required for cell proliferation and viability. This kind of antiproliferative effect occurred in both HPV-positive and HPV-negative cells. The second group of antiproliferative effects is more specific to HPV-positive cells, and it is possible this could be due to inhibition of HPV16 E7 phosphorylation.
Our data leave several questions open to future investigation. The mechanisms by which CKII inhibition leads to inhibition of cell growth are not fully understood, nor is the extent to which inhibition of E7 phosphorylation mediates the HPV-specific effects. While our data are consistent with a key role for E7 phosphorylation, the downstream effects of MRP-8/14-mediated inhibition of E7 phosphorylation are not clear. Storey et al. showed that substitution of one of the two serine residues of the E7 CKII site, Ser31 or Ser32, only slightly decreased its ability to cooperate with the EJ-ras oncogene to transform primary baby rat kidney cells (53
). Barbosa et al. also showed that mutation of one of the two serines did not show any significant biological activity (3
). However, simultaneous mutation of both serines impaired or significantly reduced its transforming activity (3
), suggesting that phosphorylation of both serines, Ser31 and Ser32, may be necessary for E7's full transforming activity.
The best-characterized E7 ligand is pRb, but the role of CKII-mediated E7 phosphorylation in its interaction with pRb and pRb-associated proteins is not understood. Using in vitro binding assays, it has previously been shown that mutation in the CKII phosphorylation site of E7 Ser 31/32 did not affect its binding to pRb (3
). However, it is not clear how well these in vitro binding assays reflect the more complex intracellular environment. Moreover, since inhibition of transformation occurred with mutation of both serines despite the lack of change in pRb binding (3
), it is also likely that the E7-pRb interaction is not the only mechanism for E7-mediated transformation or stimulation of cell cycling (6
). Consistent with this, MRP-8/14-mediated CKII inactivation and E7 hypophosphorylation in vivo may affect other pRb indirect and/or pRb-independent cell cycle regulatory pathways.
E7 phosphorylation of high-risk HPV types may be involved in E7-mediated degradation of pRB (25
). Phosphorylation of HPV16 E7 has been shown to increase the binding affinity of E7 to the basic subunit of the TFIID complex, the TATA box-binding protein (TBP), and TBP-associated factor 110 (TAF-110) protein (31
). E7 binding to F-actin requires HPV16 E7 phosphorylation (43
), and HPV16 E7 phosphorylation may play a role in E7 interaction with p53 (33
). A CKII phosphorylation-defective HPV16 E7 mutant severely impaired its ability to induce tetrasomy in primary foreskin keratinocytes, suggesting that E7 phosphorylation may be involved in induction of genetic instability (50
). Taken together, these data indicate that CKII-mediated E7 phosphorylation may be critical for its oncogenic functions and suggest that MRP-8/14-mediated inhibition of CKII may affect HPV-induced cell cycling through multiple mechanisms.
In summary, we have found that the S100 calcium-binding protein complex MRP-8/14 inhibits CKII-mediated HPV16 E7 phosphorylation in vivo and in vitro. We showed MRP expression in normal primary epithelial cells in culture, with loss of their expression in several HPV-positive cervical cancer cell lines. The mechanisms of loss of MRP-8 and -14 protein expression in tumor cells are not well understood but may occur during the multistep cell transformation process. Consistent with this, loss of MRP expression was observed in 16MT cells during the process of HPV-induced immortalization. MRP-8/14 protein expression was detected only in parental cells, and once the primary keratinocytes were immortalized with HPV16 MRP-8/14 expression was lost and CKII activity was elevated. However, absence of MRP expression in HPV-negative C33A cervical carcinoma cells indicated that inactivation of MRP expression was not specifically due to HPV infection. Activation of CKII has been reported in tumor cells of different origins (11
), and mechanisms of its activation, particularly the role of MRP-8/14 in its regulation, are not well understood. It is possible that loss of MRP expression and elevation of CKII activity in high-risk HPV-infected precancerous lesions may lead to higher levels of phosphorylation of E7, increasing E7 oncogenic activity and possibly progression of HPV-associated neoplasia.