|Home | About | Journals | Submit | Contact Us | Français|
Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKCδ by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKCδ downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKCδ at the activation loop (Thr505), turn motif (Ser643), hydrophobic motif (Ser662) and Tyr-311 sites to a greater extent in HeLa/CP cells compared to HeLa cells. Prolonged treatment with PDBu led to downregulation of PKCδ in HeLa but not in HeLa/CP cells. The PKC inhibitor Gö 6983 inhibited PDBu-induced downregulation of PKCδ, decreased Thr505 phosphorylation and increased PKCδ tyrosine phosphorylation at Tyr-311 site. However, knockdown of c-Abl, c-Src, Fyn and Lyn had little effect on PKCδ downregulation and Tyr311 phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor Ly294002 and mTOR inhibitor rapamycin restored the ability of PDBu to downregulate PKCδ in HeLa/CP cells. Knockdown of mTOR and rictor but not raptor facilitated PKCδ downregulation. Depletion of PKCε also enhanced PKCδ downregulation by PDBu. These results suggest that downregulation of PKCδ is regulated by PKCε and mammalian target of rapamycin complex 2 (mTORC2).
Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine protein kinases, plays a critical role in regulating diverse cellular processes including cell proliferation, cell survival, apoptosis, cell migration and tumor promotion . The PKC family can be categorized into three groups based on their structure, function and biochemical regulation [2–5]. Conventional PKCs (α, βI, βII and γ) require Ca2+ and diacylglycerol (DAG) for their activities. Novel PKCs (δ, ε, η and θ) are Ca2+-independent but DAG-dependent whereas atypical PKCs (ζ, λ/ι) do not require Ca2+ or DAG for their activities. PKC serves as the receptor for tumor promoting phorbol esters, which are potent activators of conventional and novel PKCs and can substitute for DAG . Prolonged treatment with tumor-promoting phorbol esters leads to degradation or downregulation of PKCs. Persistent activation or downregulation of PKCs by phorbol esters has been associated with tumor promotion . Thus, understanding the mechanism of PKC downregulation is of critical importance.
PKCs are regulated not only by cofactors but also by phosphorylation. PKCs can be phosphorylated at three conserved Ser/Thr phosphorylation sites: (i) activation loop (A-loop), (ii) turn motif (TM), and (iii) hydrophobic motif (HM) [3, 8]. The phosphorylation of PKCs primes them for activation by cofactors [3, 8]. Phosphorylation of PKCs may involve both autophosphorylation  and transphosphorylation . Most of the studies on PKC phosphorylation have been performed with conventional PKCs. It is generally believed that phosphorylation at the A-loop is mediated by phosphoinositide-dependent kinase-1 (PDK1) which has been shown to be the upstream kinase for several members of the AGC family of kinases, including PKC isozymes [11–], Akt/PKB  and p70S6K . Once phosphorylated at the A-loop, PKCs are believed to undergo autophosphorylation at the TM and the HM . However, recent studies suggest that Akt and conventional PKCs are phosphorylated at the TM and HM by the mammalian target of rapamycin complex 2 (mTORC2) [19–21].
There are controversies regarding the mechanism of phosphorylation of PKCδ at the A-loop (Thr505), TM (Ser643) and HM (Ser662). While it is generally believed PKCδ is phosphorylated at the activation loop by PDK1, it was also shown to be transphosphorylated by PKCε . Ser643 site is believed to be an autophosphorylation site whereas phosphorylation at the C-terminal hydrophobic domain of PKCδ and PKCε is believed to be regulated by rapamycin-sensitive mTOR . A recent study, however, suggests that PKCε but not PKCδ is phosphorylated at both the TM and HM sites by mTORC2 . PKCδ can also be phosphorylated at several tyrosine residues . In contrast to serine/threonine phosphorylation, which is a common regulatory mechanism for PKC isozymes, tyrosine phosphorylation is a unique regulatory mechanism for PKCδ .
It is believed that priming phosphorylation of PKCs at serine/threonine sites maintains them in a closed, protease/phosphatase resistant form [3, 8] and dephosphorylation predisposes them to downregulation [25–27]. In contrast to cPKCs, which are stabilized by phosphorylation, A-loop phosphorylation of PKCδ was shown to be necessary for phorbol ester-mediated downregulation of PKCδ . On the other hand, phosphorylation of PKCδ at Y311 was reported to be important for downregulation of PKCδ by Src but not by phorbol esters . It is now realized that the immunoreactivity of the PKCδ antibody used in these studies is altered by the PDBu treatment . In addition, some of the studies on PKCδ phosphorylation and downregulation were performed in serum-deprived adherent cells grown in suspension  and the regulation of PKCs in suspension culture could be distinct from adherent cells . Furthermore, the regulation of exogenously expressed PKCδ may be different from the endogenous PKCδ since the tight regulation of PKCδ is often lost during PKCδ overexpression . Thus, the mechanism of PKCδ downregulation is yet to be unraveled.
PKCδ plays a critical role in apoptosis [32, 33]. It is believed that proteolytic cleavage of PKCδ is essential for the induction of apoptosis. PKCδ is also a positive regulator of cell growth. It can function as an antiapoptotic protein and can contribute to cell transformation and metastasis [1, 7, 33, 34]. We have shown that acquisition of resistance by HeLa cells to the chemotherapeutic drug cisplatin was associated with loss of regulation of PKCδ [35, 36]. The ability of phorbol esters to downregulate PKCδ was compromised in cisplatin-resistant HeLa cells . Thus, this system provides a unique tool to understand the mechanism of PKCδ downregulation by PKC activators. Our results show that downregulation of PKCδ is regulated by PKCε and mTORC2.
PDBu was purchased from LC Service Corporation (Woburn, MA). Polyclonal antibodies to tubulin, PKCδ and PKCε, and monoclonal antibody to GAPDH were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibody to PKCα was from Upstate Biotechnology, Inc. (Lake Placid, NY) and monoclonal antibody to PKCδ was from BD Transduction Laboratories (San Jose, CA). Polyclonal antibodies to phospho-PKCδ were obtained from Cell Signaling (Beverly, MA). Non-targeting siRNA and siRNA SMARTpool against PKCα, -ε, c-Src, Fyn, Lyn, c-Abl, PDK1, raptor, rictor and mTOR were obtained from Dharmacon (Lafayette, CO). Horseradish peroxidase conjugated goat anti-mouse and donkey anti-rabbit antibodies were obtained from JacksonImmunoResearch Lab. Inc. (West Grove, PA). Poly(vinylidenedifluoride) membrane was from Millipore (Bedford, MA) and enhanced chemiluminescence detection kit was from Amersham (Arlington Heights, IL).
Human cervical carcinoma HeLa cells and its cisplatin-resistant variants (HeLa/CP) were maintained in Dulbecco's modified minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine, and kept in a humidified incubator at 370C with 95% air and 5% CO2. HeLa/CP cells were maintained in drug-free media.
Equivalent amounts of proteins were electrophoresed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred electrophoretically to poly(vinylidene difluoride) membrane. Immunoblot analyses were performed as described before .
Control non-targeting siRNA or SMARTpool siRNA against PKC isozymes, Src family tyrosine kinase, rictor, raptor or mTOR were introduced into HeLa or HeLa/CP cells using Lipofectamine 2000 (Invitrogen) and manufacturer’s protocol. Briefly, cells were seeded one day before transfection. 48 h following siRNA transfection, cells were treated as indicated in the text and processed for Western blot analysis as described before [36, 38].
We have shown previously that downregulation of PKCδ by PDBu is compromised in HeLa cells that acquired resistance to the anticancer drug cisplatin (HeLa/CP) [35, 36]. Since phosphorylation status of PKCs regulates their stability, we compared the effect of PDBu on the phosphorylation of PKCδ at the Ser/Thr and Tyr sites. Figure 1 shows that PDBu caused an increase in phosphorylation of PKCδ at the A-loop (T505), TM (S643), HM (S662) and Y-311 sites. The levels of phospho-PKCδ at all of these sites were greater in HeLa/CP cells compared to HeLa cells.
Since chronic treatment with PKC activators results in PKCδ downregulation, we examined the effects of several PKC inhibitors on PKCδ downregulation induced by PDBu. While Gö 6983 inhibits all PKCs, Gö 6976 inhibits conventional PKCα and -βI  and rottlerin inhibits PKCδ  although it has many other targets. Figure 2 shows that PDBu induced substantial downregulation of PKCδ in HeLa cells but not in HeLa/CP cells whereas PDBu led to downregulation of PKCα in both HeLa and HeLa/CP cells. Both Gö 6976 and Gö 6983 prevented PKCδ downregulation by PDBu in HeLa cells although Gö 6976 was less effective than Gö 6983. In contrast, rottlerin had little effect on PDBu-induced downregulation of PKCδ. These results suggest that PKCδ downregulation is primarily regulated by novel PKCs.
It has been reported that phosphorylation of PKCδ at T505 is important for PKCδ downregulation . We therefore examined the effects of PKC inhibitors on PDBu-induced phosphorylation of PKCδ at T505 site. Figure 3 shows a brief treatment with PDBu induced phosphorylation of PKCδ at T505 site. The general PKC inhibitor Gö 6983 inhibited PDBu-induced phosphorylation of PKCδ at T505 whereas Gö 6976 or rottlerin had no effect. Since atypical PKCs are phorbol ester-insensitive, this is consistent with an earlier report that PKCδ is transphosphorylated at the T505 site by PKCε . None of the inhibitors prevented phosphorylation of PKCδ at S643 site. PDBu caused a modest increase in PKCδ level when detected using the polyclonal antibody to PKCδ. However, PKCδ level in PDBu-treated cells was substantially less when monoclonal antibody from BD Transduction Laboratories was used to detect PKCδ. This decrease in PKCδ was blocked by Gö 6983 but not by Gö 6976 or rottlerin. In fact, PKCδ level detected using either monoclonal or polyclonal antibody to PKCδ was equivalent in cells treated with Gö 6983. These results suggest that the immunoreactivity of monoclonal antibody to PKCδ is decreased when PKCδ is phosphorylated at T505 site as has been reported .
Since PKCδ is not only regulated by Ser/Thr phosphorylation but also by tyrosine phosphorylation, we examined the effects of PKC inhibitors on PDBu-induced PKCδ tyrosine phosphorylation. As shown in Figure 3, Gö 6983 caused a substantial increase in PDBu-induced PKCδ Y311 phosphorylation; Gö 6976 also caused a modest increase whereas rottlerin had only a little effect on Y311 phosphorylation. Thus, an increase in PKCδ Y311 phosphorylation appears to correlate with inhibition of PKCδ downregulation by these PKC inhibitors (Fig. 2 vs. Fig. 3).
It is believed that phosphorylation of PKCδ at Y311 is mediated by Src family tyrosine kinases (SFKs) [24, 29, 41]. Since PDBu failed to induce substantial downregulation of PKCδ in HeLa/CP cells, we depleted several SFKs, including c-Src, Fyn and Lyn using siRNA and examined the effect of SFK knockdown on PKCδ downregulation. A recent report suggested that c-Abl rather than c-Src phosphorylates PKCδ at Y311 site (42). Therefore, we also examined the effect of c-Abl knockdown on PKCδ downregulation by PDBu. As shown in Figure 4, specific knockdown of c-Abl and SFKs was achieved using siRNA although depletion of these kinases had little effect on PKCδ downregulation and Y-311 phosphorylation.
It has been reported that PKCε regulates PKCδ phosphorylation at T505 site via Src-dependent PKCδ tyrosine phosphorylation [22, 41]. We therefore examined if depletion of PKCε affects PKCδ phosphorylation at Y311 and T505 sites. Figure 5 shows that siRNA silencing of PKCε attenuated PDBu-induced Y311 phosphorylation in HeLa cells. Knockdown of PKCε had little effect on the phosphorylation of PKCδ at T505 site when treated with PDBu for 30 min. However, when cells were treated with PDBu for 4 h and 18 h, the phosphorylation of PKCδ at T505 site was decreased in PKCε-depleted cells. The levels of PKCδ following treatment with PDBu for 4 h and 18 h were also less in PKCε-depleted cells compared to control siRNA-transfected cells. Thus, depletion of PKCε enhanced PKCδ downregulation and this was associated with decrease in PKCδ Y311 phosphorylation.
It is generally believed that phosphorylation of PKCδ at the activation loop is mediated by PDK1  whereas phosphorylation at the hydrophobic site is mediated by rapamycin-sensitive mTOR . To determine the importance of Ser/Thr phosphorylation on PKCδ downregulation, we examined the effect of Ly294002, a pharmacological inhibitor of PI3-K/PDK1 and rapamycin, an inhibitor of mTOR on PKCδ downregulation in HeLa/CP cells. Figure 6A shows that prolonged treatment with PDBu caused only a modest decrease in PKCδ in HeLa/CP cells although PDBu caused substantial downregulation of PKCα and PKCε. Pretreatment with Ly294002 and rapamycin restored the ability of PDBu to downregulate PKCδ. These inhibitors also enhanced downregulation of PKCε and PKCα by PDBu (Fig. 6A). PKCδ was phosphorylated at both Ser/Thr and Tyr residues and PDBu caused a substantial increase in Y311 phosphorylation, which was retained even when HeLa/CP cells were treated with PDBu for 16 h. Brief treatment with PDBu for 10 min or 30 min also caused a modest increase in PKCδ phosphorylation at Ser/Thr sites. Pretreatment with Ly294002 and rapamycin for a prolonged period had little effect on T505 phosphorylation but it attenuated phosphorylation of PKCδ at Y311, S643 and S662 sites by PDBu. Thus, dephosphorylation of PKCδ at both Ser and Tyr residues was associated with PKCδ downregulation.
To further rule out the involvement of PDK1 in mediating phosphorylation of PKCδ at T505, we depleted PDK1 using siRNA. Knockdown of PDK1 failed to inhibit T505 phosphorylation or enhance downregulation of PKCδ by PDBu (Fig. 6B). Thus, the effect of Ly294002 on PKCδ downregulation was not due to inhibition of PDK1.
mTOR can complex with either raptor or rictor to form mTORC1 and mTORC2, respectively . Since rapamycin enhanced PKCδ downregulation, we compared the effects of knockdown of rictor, raptor or mTOR on PDBu-induced downregulation of PKCδ in HeLa/CP cells. As shown in Figure 7, knockdown of rictor and mTOR but not raptor facilitated PKCδ downregulation by PDBu. This downregulation was accompanied by a decrease in phosphorylation at Y311, S643 and S662 sites.
It has been reported that the phosphorylation of PKCε at the TM and HM sites is regulated by mTORC2 . We therefore compared the effect of depletion of PKCε and rictor on PKCδ downregulation in HeLa/CP cells. Figure 8 shows that silencing of either PKCε or rictor by siRNA enhanced PKCδ downregulation by PDBu and the effect was greater when both PKCε and rictor were depleted. To determine if there was any association between phosphorylation of PKCδ at a particular site and PKCδ downregulation, we monitored PKCδ phosphorylation following a brief treatment (10 min) with PDBu. Depletion of either PKCε or rictor caused a modest decrease in Y311 and S643 phosphorylation but knockdown of both PKCε and rictor caused a substantial decrease in Y311, S643 and S662 phosphorylation. These results suggest that dephosphorylation at both Ser and Tyr sites may be important for PKCδ downregulation.
Long-term cellular responses, such as cell proliferation, cell death and tumor promotion activities of PKCδ have been linked with PKCδ downregulation . The dephosphorylation of priming sites of PKCα and PKCε has been shown to be important for their downregulation [25–27]. Phosphorylation of cPKC at TM is important for protein maturation and stability [19, 20]. mTORC2 has been shown to regulate TM and HM phosphorylation of PKCα and PKCε but not of PKCδ . The results of our present study demonstrate that downregulation of PKCδ by PDBu is also regulated by mTORC2. We have also made a novel observation that PKCε regulates activation-induced downregulation of PKCδ. Furthermore, dephosphorylation of PKCδ at both Ser/Thr and Tyr residues appears to be important for PKCδ downregulation.
In contrast to conventional PKCα or novel PKCε, phosphorylation of PKCδ at the activation loop (T505) is believed to be required for its dowregulation because the Ser/Thr phosphatase inhibitor calyculin A increased T505 phosphorylation and enhanced TPA-induced degradation of PKCδ . We have also found that Gö 6983 inhibited T505 phosphorylation and blocked PKCδ downregulation (Fig. 2). However, Gö 6983 also caused a substantial increase in phosphorylation of PKCδ at Y311 site. In addition, it is now realized that the monoclonal antibody from BD Transduction laboratory does not recognize PKCδ phosphorylated at T505 site . Even a brief treatment with PDBu that did not decrease PKCδ level detected using the polyclonal antibody to PKCδ in fact caused substantial decrease in PKCδ when detected using the monoclonal antibody to PKCδ (Fig. 3). This decrease in PKCδ level detected using monoclonal PKCδ antibody was reversed when T505 phosphorylation was inhibited by Gö 6983. Since calyculin A inhibits dephosphorylation of PKCδ at T505 site, it increases T505 phosphorylation in PDBu-treated cells. Because the monoclonal antibody does not detect T505 PKCδ, a decrease in PKCδ in calyculin A-treated cells in the earlier study could be explained by the loss of immunoreactivity of monoclonal PKCδ antibody towards T505 PKCδ rather than enhanced PKCδ downregulation.
PKCδ is phosphorylated not only at the Ser/Thr sites but also at Tyr residues . It has been reported that phosphorylation of PKCδ at Y311 is important for downregulation of PKCδ by activated Src but not by phorbol esters . Our results suggest that dephosphorylation of PKCδ at Y311 is associated with PDBu-induced PKCδ downregulation. First, PKC inhibitors that inhibited PKCδ downregulation by PDBu increased Y311 phosphorylation (Fig. 3). Gö 6983 caused a substantial increase in tyrosine phosphorylation of PKCδ at Y311 site. The cPKC inhibitor Gö 6976 also enhanced Y311 phosphorylation albeit less effectively than general PKC inhibitors. The ability of these inhibitors to induce PKCδ tyrosine phosphorylation correlated with their ability to prevent PKCδ downregulation. Second, the lack of PKCδ downregulation by PDBu in cisplatin-resistant HeLa cells was associated with increase in PKCδ tyrosine phosphorylation. Third, Ly294002 and rapamycin resored PKCδ downregulation in HeLa/CP cells and attenuated PDBu-induced phosphorylation of PKCδ at Y311 site (Fig. 6A). Finally, knockdown of PKCε and rictor by siRNA decreased PDBu-induced Y311 phosphorylation and enhanced PKCδ downregulation (Fig. 5, Fig. 7 and Fig. 8).
It is generally believed that PKCδ is phosphorylated at Y311 site by Src family tyrosine kinases [24, 29, 43]. However, knockdown of c-Src did not inhibit PDBu-induced PKCδ Y311 phosphorylation in HeLa/CP cells (Fig. 4), suggesting that phosphorylation at Y311 by PDBu may be mediated by a tyrosine kinase distinct from c-Src. Several Src family tyrosine kinases have been shown to complex with PKCδ . It has been reported that Y311 phosphorylation in response to H2O2 is mediated by c-Abl and not by Src kinases . However, knockdown of c-Abl, c-Src, Fyn or Lyn had little effect on Y311 phosphorylation and PKCδ downregulation in HeLa/CP cells (Fig. 4B). Most of the studies on PKCδ tyrosine phosphorylation have been performed in response to H2O2. It has been reported that PMA does not activate SFK activity directly and the identity of phorbol ester-activated kinase that phosphorylates PKCδ at Y311 remains to be determined .
It has been reported that there is a cross-talk between PKCδ and PKCε. There is, however, controversy regarding how PKCε regulates PKCδ phosphorylation. PKCδ protein expression and T505 phosphorylation were increased in PKCε−/− mice compared to wild-type mice . In contrast, adenoviral delivery of PKCε in cardiomyocytes increased phosphorylation of PKCδ at T505 and Y311 sites . However, the heterologously expressed PKCδ was not influenced by PKCε overexpression and the investigators questioned the suitability of studies using PKC overexpression. We have used siRNA silencing rather than PKCε overexpression to investigate the involvement of PKCε on PKCδ downregulation. We have found that depletion of PKCε enhanced PDBu-induced downregulation of PKCδ in both HeLa and HeLa/CP cells (Fig. 5 and Fig. 8). We were unable to detect a consistent decrease in T505 phosphorylation by PKCε depletion although knockdown of PKCε did attenuate PDBu-induced phosphorylation of PKCδ at Y311 (Fig. 5 and Fig. 8). Neither Ly294002 nor PDK1 depletion affected phosphorylation of PKCδ at T505 site. The observations that Gö 6983 inhibited T505 phosphorylation (Fig. 3) and DN-PKCδ was dephosphorylated at T505 site (data not shown), suggest that PKCδ is autophosphorylated at T505 site.
Although Ly294002 did not inhibit phosphorylation of PKCδ at T505 site, it potentiated PKCδ downregulation by PDBu (Fig. 6). Ly294002 may influence PKCδ downregulation via PKCε, which is also believed to be regulated by the PI3-K pathway [3, 23]. In addition, PI3-K acts upstream of mTOR, which has been implicated in regulating phosphorylation of PKCδ at the hydrophobic site . The earlier studies have focused on rapamycin-sensitive mTOR or mTORC1 . We have found that brief treatment with Ly294002 or rapamycin had no effect on the phosphorylation of PKCδ at the A-loop, TM, HM or Y-311 sites (data not shown). However, prolonged treatment with Ly294002 or rapamycin decreased phosphorylation of PKCδ not only at the S662 site but also at S643 and Y311 sites. It has been reported that prolonged rapamycin treatment could inhibit the assembly of mTORC2 . Thus, the effects of Ly294002 and rapamycin may be mediated via mTORC2 rather than mTORC1. Our results show that knockdown of rictor and mTOR but not raptor restored PDBu-induced downregulation of PKCδ in HeLa/CP cells (Fig. 7), suggesting the importance of mTORC2 in regulating PKCδ downregulation. Interestingly, knockdown of rictor not only caused a decrease in phosphorylation of PKCδ at the TM but also decreased Y311 phosphorylation.
We have found that an increase in PKCδ downregulation by knockdown of either PKCε or rictor was associated with a decrease in both Y311 and S643 phosphorylation. Depletion of both PKCε and rictor caused a substantial decrease in phosphorylation of PKCδ at S643, S662 and Y311 sites, and this was associated with a dramatic increase in PDBu-induced downregulation of PKCδ in HeLa/CP cells (Fig. 8). Since the tight regulation of PKC is lost when overexpressed, it is difficult to attribute a particular site responsible for PKCδ downregulation. Furthermore, phorbol ester has been shown to phosphorylate PKCδ at several newly identified Ser/Thr sites, including S299 and S304 at the V3 region (37) and the involvement of these sites on PKCδ downregulation remains to be established.
While mTORC2 is known to regulate phosphorylation of Akt, cPKCs and PKCε at the TM and HM sites [19, 20], we have found that Ly294002, rapamycin and rictor knockdown not only decreased phosphorylation at these sites but also attenuated phosphorylation at Y311 by PDBu (Fig. 6 and Fig. 8). It is conceivable that rictor regulates PKCδ Y311 phosphorylation via PKCε, which is a binding partner of Src (47). Although knockdown of Src did not decrease Y311 phosphorylation, it is possible that PKCε also interacts with a related tyrosine kinase, which phosphorylates Y311 in response to PDBu. While future studies should discern how rictor and PKCε regulate phosphorylation of PKCδ at the Ser/Thr or Tyr sites, the present study provides evidence that like cPKCα and nPKCε, the stability of PKCδ is also regulated by mTORC2. Furthermore, an interplay between PKCε, mTORC2 and perhaps a member of the SFK regulates downregulation of PKCδ by PDBu.
This work was supported by the grant CA071727 from the National Cancer Institute.
The authors wish to thank Drs. Baohua Sun and Giridhar Akkaraju for cloning of PKCδ, Jie Huang, Haidi Tu and Chandreyi Basu for technical assistance, and Deepanwita Pal, Dr. Eswar Swamy and Dr. Qiang Zeng for help with the manuscript.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.