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First published online 23 June 2009
doi: 10.1242/jcs.046979
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Research Article |
1 Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL 33136, USA
2 CIEMAT, 28040 Madrid, Spain
3 University of Bonn Institute of Physiological Chemistry, 53115 Bonn, Germany
4 Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA
* Author for correspondence (e-mail: psalas{at}miami.edu)
Accepted 14 April 2009
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) is a key organizer of cellular asymmetry. Sequential extractions of intestinal cells showed a pool of enzymatically active PKC and the chaperone Hsp70.1 attached to the apical cytoskeleton. Pull-down experiments using purified and recombinant proteins showed a complex of Hsp70 and atypical PKC on filamentous keratins. Transgenic animals overexpressing keratin 8 displayed delocalization of Hsp70 and atypical PKC. Two different keratin-null mouse models, as well as keratin-8 knockdown cells in tissue culture, also showed redistribution of Hsp70 and a sharp decrease in the active form of atypical PKC, which was also reduced by Hsp70 knockdown. An in-vitro turn motif rephosphorylation assay indicated that PKC is dephosphorylated by prolonged activity. The Triton-soluble fraction could rephosphorylate PKC only when supplemented with the cytoskeletal pellet or filamentous highly purified keratins, a function abolished by immunodepletion of Hsp70 but rescued by recombinant Hsp70. We conclude that both filamentous keratins and Hsp70 are required for the rescue rephosphorylation of mature atypical PKC, regulating the subcellular distribution and steady-state levels of active PKC.
Key words: PKC, Keratins, Epithelial polarity, Protein chaperones, Apical kinases
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Introduction |
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(iota, /
; one of the two atypical isoforms, which are collectively referred to as aPKC) is an oncogene product causative of lung non-small cell carcinoma and is a predisposing factor for colon cancer when overexpressed (Fields and Regala, 2007
). It is also a key regulator of asymmetry in various tissues, and is conserved in evolution. It was originally identified as such in C. elegans along with its partners (partition deficient, PAR) PAR3 and PAR6 (Rose and Kemphues, 1998). In neurons, PKC controls axonal polarity under extrinsic Wnt signaling (Zhang et al., 2007). In epithelial cells, the PKC-PAR3-PAR6 complex is associated with tight junctions and generates a signaling gradient that specifies the apical domain and the localization of the junctions. The tight-junctional scaffold for this complex includes Crumbs, Pals1 and PatJ (Suzuki and Ohno, 2006). The functionality of the PKC kinase domain depends on its conformation, controlled by phosphorylations in the activation loop (T403 in PKC) by PDK-1 and in the turn motif (T555 in PKC) (Newton, 2003).
Unlike other kinases, PKC isozymes lose their activation phosphorylation and conformation in a substrate-dependent manner (Dutil et al., 1994), i.e. they become inactivated as a consequence of their own function. This characteristic has been used to inactivate conventional and novel PKC by prolonged treatment with phorbol esters (Savart et al., 1992; Hansra et al., 1999; Lang et al., 2004). The loss of PKC conformation is controlled by phosphatases and followed by attachment to still-unidentified cytoskeletal elements in the Triton-insoluble fraction (Gao et al., 2008). Once dephosphorylated, the PKC molecules are ubiquitinylated and degraded (Chen et al., 2007). Hsp70 chaperone proteins (Hsp70.1 hereafter referred to as Hsp70, and the constitutively expressed Hsc70) (Daugaard et al., 2007) bind dephosphorylated PKC (including 
, β and the atypical 
isoforms) (Gao and Newton, 2002) and rescue them from degradation to regain function (Gao et al., 2006). The activation loop and turn motif are rephosphorylated, although it is unclear whether PDK-1 or other kinases are involved in the process. This mechanism effectively extends the active life of PKC (Newton, 2003) and, conceivably, can regulate the overall steady-state cellular levels of the kinase.
A role of Hsp70 in rescuing dephosphorylated atypical PKC isoform has not yet been reported. However, based on the structural conservation of the PKC catalytic domain and the presence of the invariant Leu554 motif (NFDSQFTNEPVQL*TPDDDDI) necessary for Hsp70 binding (Gao and Newton, 2006), it is reasonable to hypothesize that Hsp70 interaction with atypical PKC is also involved in maintaining the functional pool of this kinase. Phosphorylation in the turn motif (T555) abrogates Hsp70 interaction (Gao and Newton, 2002), thus making this phosphorylation an ideal marker for the active-conformation PKC pool that cannot interact with Hsp70. To our knowledge, no attempts have been made to identify the specific cytoskeletal proteins that bind PKC in the inactive conformation. However, various chaperones, including Hsp70 involved in PKC rescue, are bound to keratin intermediate filaments (IFs) (Liao et al., 1995; van den Ijssel et al., 1999; Planko et al., 2007). IFs are formed by family of cytoskeletal proteins, encoded by approximately 60 different genes in mammals. Mutations identified in IF proteins responsible for human disease outnumber the pathogenic mutations in both actins and tubulins. In some cases, these mutations do not abrogate filament polymerization or structural integrity (Omary et al., 2004), and, therefore it is unlikely that the phenotype results from mechanical weakness of the cells. An emerging body of evidence suggests that IFs have non-mechanical functions (Oriolo et al., 2007a), including antiapoptotic activity (Marceau et al., 2007) related to intracellular signaling, but mechanistic explanations for those functions are still missing (Magin et al., 2007; Toivola et al., 2005).
In addition, we reported a broad loss of the apical domain phenotype in the villus enterocytes of keratin 8 (K8)-null mice. Yet, a molecular model for that phenotype has been elusive (Ameen et al., 2001). Together, these facts led us to hypothesize that a pool of PKC might be associated with a keratin scaffold in epithelial cells. The results not only corroborated the existence of a pool of keratin-bound PKC, but also showed a novel function of the cytoskeleton in the post-translational regulation of PKC expression and localization.
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co-distributes and co-purifies with the keratin cytoskeleton). Like in other epithelial cells (Suzuki and Ohno, 2006), XZ confocal sections showed PKC distributed only in the apical domain of CACO-2 cells, colocalizing with PAR3 only in the apical-most part of the cell-cell contacts (Fig. 1A,B). In the rest of the apical region, away from tight junctions, it colocalized with PAR6 (not shown), along with keratin IFs (Fig. 1B,C), but not with PAR3. Triton-100-insoluble PKC (Fig. 1C, green) colocalized with the apical keratin signal (Fig. 1C, red). PAR3 was not detected in the Triton X-100 extracted monolayers (not shown). To characterize the cytoskeletal association of PKC and its partners, we used an extraction method modified from a well-established method to isolate keratins that dissects actin. The Triton X-100 soluble supernatant (S1) contains cytosolic and membrane proteins. The Triton-insoluble (cytoskeletal) pellet was further extracted in 1.5 M KCl, and separated into supernatant (S2) and pellet (P). Together, the three fractions contained the entire set of cellular proteins. Actin was present in S1 and S2, but not in P (Fig. 1D). Keratins were found almost exclusively in P (Fig. 1D). PKC and pT555 PKC were present in all three fractions. It was surprising to find pT555 PKC in the P fraction because previous publications indicated that only the non-phosphorylated inactive forms of PKC bind to the cytoskeleton (Gao et al., 2008). Well-known PKC partners and components of the conventional tight-junction scaffold that binds PKC, including PAR3, PAR6, 14-3-3 (PAR5 homolog), ZO-1 and PALS1 (Suzuki and Ohno, 2006), were not present in the keratin pellet P, but were found in S1 or S2. Hsp70 proteins, however, were present in all three fractions (Fig. 1D). Protein partitioning in lipid rafts represents an exception to the Triton insolubility criterion of cytoskeletal association. We ruled out raft association of the Triton-insoluble PKC by lack of co-purification with the ganglioside GM1, found only in S1, and by lack of effect of cholesterol sequestering agents filipin III and β-cyclodextrin (CD) (supplementary material Fig. S1).
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in the three different fractions was active kinase we used a standard PKC activity assay that is not isoform-specific. Thus, although PKC is expressed as the prevalent isoform in CACO-2 cells (Wald et al., 2008), it was expected that other PKC isoforms would contribute to the activity as well. To dissect PKC activity from conventional and novel PKC activities, we used inhibitors that preferentially block atypical PKC (RO-31-8220) or conventional and novel PKCs (GF10923X). A PKA inhibitor (H-89) was used to control for possible non-specificity of the assay. Substantial PKC activity was found in S1, S2 and P fractions (Fig. 2A). Importantly, PKC activity in the P fraction was only blocked by RO-31-8220, indicating that it is due to atypical PKC (Fig. 2A). More direct evidence was obtained by shRNA knockdown of PKC by lentiviral transduction. It was very effective (>90%), with only a small amount of PKC remaining in the S1 fraction (Fig. 2C). When the same fractions were assayed for PKC activity, PKC knockdown abrogated PKC activity in the keratin pellet P. It is of note that all the results in Figs 1 and 2 were normalized by protein amount, but the size of each fraction, in terms of total cellular protein was very different. S1 comprised 75% of the total cellular protein and P only 7% (Table 1). Accordingly, the relative amounts of pT555-PKC signal and PKC activities were proportional to the size of the corresponding fraction (Table 1).
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PKC and Hsp70 form a ternary complex with intermediate filaments in vitro
To establish whether PKC binds directly to keratins, we expressed V5-6xHis-tagged PKC in HEK293T cells and purified it on Ni2+ columns under non-denaturing conditions. The desalted eluates containing PKC but not endogenous Hsc70 (Fig. 3A) were used to overlay blots of highly purified recombinant keratins (K8, K18 and K19). We found direct binding of PKC (V5 signal) to K8. However, no pT555 signal was detected despite the fact that a fraction of the PKC in the input was phosphorylated in T555 (Fig. 3A,B), suggesting that only the dephosphorylated-T555 (inactive conformation) PKC binds to K8.
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), next we tested the interaction of PKC with Hsp70. Surprisingly, we did not find any Hsp70 co-immunoprecipitation with PKC in the soluble fraction (S1) of CACO-2 cells (Fig. 3C). Separately, both Hsp70 (not shown) and PKC (Fig. 3D, pull-down, IF) bound directly to highly purified intermediate filaments from CACO-2 cells in a pull-down assay. The direct interaction of PKC with filamentous keratins confirmed the results from overlay assays described above. However, the addition of Hsp70 to the PKC-keratin pull-down mix increased the amount of PKC bound to intermediate filaments threefold (Fig. 3D,E), showing the existence of a ternary complex of filamentous keratins, Hsp70 and PKC. Similarly to the results from overlay assays, we did not detect any pT555 signal in the PKC bound directly to intermediate filaments or in the additional PKC bound to the filaments in the presence of Hsp70 (Fig. 3D, pull-down, pT555), albeit the input did contain pT555 PKC. This result indicates that only the pT555-dephosphorylated form of PKC interacts with Hsp70 in a ternary complex with IF. This is consistent with the binding of another atypical isoform, PKC, to Hsp70 that occurs only when the turn motif is dephosphorylated (Gao and Newton, 2002).
Keratins and Hsp70 post-translationally regulate steady state levels of PKC
Hsp70 and Hsc70 are products of two different genes that display 86% identity at the amino acid level. Hsc70 is constitutively expressed whereas Hsp70 is expressed only under stress in normal tissues (Daugaard et al., 2007) such as intestinal epithelium (Baumler et al., 2007). However, in some tissue culture cells, including CACO-2 cells, both Hsp70 and Hsc70 are constitutively expressed (Hirasaka et al., 2008; Broquet et al., 2003), albeit Hsp70 expression can be further upregulated by stress (Pierzchalski et al., 2008). Importantly, whereas it is unclear to what extent the functions of both proteins are redundant, Hsp70 and Hsc70 share several clients and a substantial level of functional overlap has been demonstrated (Singh et al., 2008; Freeman and Morimoto, 1996; Haag Breese et al., 2006).
To understand the possible biological implications of a PKC-Hsp70 interaction based on filamentous keratins, we started by knocking down IFs using lentiviral-mediated expression of anti-K8 shRNA. This is possible because differentiated CACO-2 cells express only one type-II keratin (K8; see two-dimensional gel analysis in supplementary material Fig. S2A), which is necessary to form the obligate heterodimers of a type-II and a type-I keratin. As in previous similar experiments (Wald et al., 2008), the knockdown was efficient, although some cells still expressed filaments (Fig. 4A, dark blue). Cells in which IF knockdown was very effective showed no PKC, except for some remnant signal in the tight-junction region (Fig. 4A, green). Both Hsp70 and Hsc70 were modestly decreased and delocalized (Fig. 4A, red). Immunoblot analysis of total extracts confirmed the K8 knockdown, and showed a modest reduction in Hsp and/or Hsc70, an almost 70% reduction in total PKC, and extensive loss (>90%) of the pT555 PKC signal (Fig. 4B,C). After testing several sequences, we have found only one shRNA sequence that effectively knocks down K8 in CACO-2 cells. The lack of a second independent shRNA sequence carried a small risk of observing `off-target' effects. However, having observed a similar decrease of PKC in K8-null mouse enterocytes (see Fig. 7L), we can reasonably rule out that possibility for the PKC readout. The reduction in total PKC was clearly much larger than the size of the PKC pool that co-purifies with IFs (Table 1), suggesting that the interaction of PKC with IF-Hsp70 is necessary to maintain a population of PKC molecules that is normally not attached to IFs.
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steady state levels were transcriptionally regulated, we measured PKC mRNA levels by qPCR. Triplicate determinations (normalized to GAPDH) yielded 1.25±0.01 (control) and 1.28±0.02 (K8 knockdown), showing that K8 knockdown did not significantly affect steady-state levels of PKC mRNA. By contrast, incubation of CACO-2 cells with the proteasome inhibitor MG-132 for only 6 hours resulted in a threefold increase in the steady-state amount of PKC (along with a tenfold increase in total protein ubiquitinylation) (supplementary material Fig. S3), indicating that, like in other cells (Gao and Newton, 2006), PKC turnover is very high. Along with the lack of transcriptional effects shown before, this result highlighted the likelihood of post-translational mechanisms effectively maintaining PKC steady-state levels.
Because of the protective role of Hsp70 in PKC regulation, it was important to test the prediction that Hsp70-mediated rescue is involved in the maintenance of normal steady-state levels of PKC. In CACO-2 cells, two members of the Hsp70 family, Hsp70 and Hsc70, are expressed in similar amounts because Hsp70 expression is leaky (Broquet et al., 2003; Liu et al., 2003). Accordingly, two different shRNA sequences were designed to knockdown Hsc70 (shRNA1) or Hsp70 (shRNA2) and were delivered by lentiviral transduction followed by puromycin selection. Both shRNAs knocked down Hsp70 and Hsc70 (Fig. 5A), possibly because of the very high level of sequence homology between the two mRNAs (>90%). Both shRNAs had no effect on other proteins, such as actin or K8, which were used as load controls (Fig. 5A). However, there was a 60-70% decrease in total PKC levels. More importantly, knockdown of Hsp and/or Hsc70 proteins with both shRNAs abrogated pT555 PKC signal (Fig. 5A,B). Because knockdown of Hsp70 proteins results in apoptosis (Aghdassi et al., 2007), all these experiments were performed within 48 hours of the lentiviral infection. Within this time window there was no increase in annexin V binding, although longer times of Hsp70 knockdown resulted in massive cell death (not shown). Additional internal controls for lack of caspase-3 activation and cleavage were also performed (Fig. 5C). Similar Hsc and/or Hsp70 knockdown experiments were analyzed by immunofluorescence and confocal microscopy. They confirmed that the knockdown of Hsp and/or Hsc70 proteins was extensive in the majority of the cells (Fig. 5D,E), and indicated that Hsp70 knockdown did not affect the expression or localization of IFs (Fig. 5F,G, also see Fig. 5A), but abrogated the pT555 PKC signal (Fig. 5H,I).
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Hsp70 is involved in facilitating the correct conformation of many proteins, so it is likely that several client kinases might be affected. Likewise, the effect of K8 knockdown could be a general phenomenon affecting several kinases. To test this idea we run a screen of 12 different kinases in a commercial facility that uses a panel of validated, normalized antibodies for immunoblot. The pattern of relative kinase expression and activation in control cells (Table 2) was identical to those published before from various pools of cell extracts [see table 1, 8 days, in Wald et al. (Wald et al., 2008)], showing the consistency of the method from one set of experiments to another analyzed at the same facility. Comparing extracts from CACO-2 cell cultures transduced with either anti-K8 shRNA lentivirus or mock viral particles, we independently confirmed the decrease in PKC (Table 2; Fig. 4B,C). Apart from this kinase, of five known Hsp70 clients, one did not change (Raf-1), whereas three showed a steep decrease (PKCβII, PKC and Akt) and another was only mildly affected (Chk-1). Interestingly, most PKC isoforms decreased, with the exception of PKC. By contrast, the PKA catalytic subunit actually showed an almost twofold increase (Table 2). Importantly, PDK-1, which is known to phosphorylate newly synthesized PKC was not affected by K8 knockdown. Because of the consistency of these measurements, it seems safe to rely on the results that show no change. However, the screen does not provide any measurement of variability, such as standard deviation. Bearing this limitation in mind, it seems safe to conclude that the phenomenon described here might affect other kinases, but is not generalized to all kinases, or even to all Hsp70 clients. To statistically confirm the significance and extend the observation to cells in vivo, we compared the levels of two Hsp70 clients in small intestine villous enterocytes of K8-null mice with control littermate animals. Thus, we could confirm that the differences shown by the kinase screen in CACO-2 cells were reproducible in the animal model and statistically significant for the other atypical PKC isoform PKC (pT410) and for pT308 Akt (Table 3), two known Hsp70 clients (Gao and Newton, 2002).
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To confirm our results in vivo, we used two mouse knockout models. The K19-null, K18+/– and hK18 R89C (knock-in) mice (K18R89C animals) have been shown to lack IFs in the intestinal crypts, where keratins form small, non-filamentous aggregates (Hesse et al., 2007). In normal littermates of K18R89C mice, the small intestinal crypts showed apical layers of Hsc70 and pT555 PKC (Fig. 6A,G). By contrast, in K18R89C transgenic mice the apical localization of Hsc70 and pT555 PKC was abrogated along with the expression of IFs (Fig. 6C,D,I,J), with the signals close to those in antibody negative controls (Fig. 6E,F,K,L). In the same animals, the expression and distribution of Hsc70 and pT555 PKC were normal in the villi (not shown). The second knockout model we used, K8-null mice, lack intermediate filaments in the villus, but display normal filaments in the crypts, because of the expression of redundant K7 (Baribault et al., 1994; Ameen et al., 2001). Accordingly, in K8-null mice the apical layer of Hsc70 was not observable in villus enterocytes (Fig. 7C). The pT555 PKC signal in villus enterocytes from control littermates was present only in the tight-junctional compartment (Fig. 7G, arrows). In K8-null mice, the IFs disappear after enterocytes move over the crypt-villus boundary (Ameen et al., 2001; Wald et al., 2008). Unlike in control animals, K8-null mice displayed the pT555 PKC signal associated with tight junctions only until the mid-villus region (Fig. 7I, arrows), not reaching the tip of the villus (Fig. 7I, arrows) as in controls. This suggests that even the tight-junction pool of PKC needs IFs to subsist.
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To validate these observations at the biochemical level, villus enterocytes were isolated from the mucosa of control and K8-null mice and analyzed by immunoblot. The intensity of Hsc70 signal was very variable among all the animals, but it was not significantly decreased in K8-null mice. The average actually showed a modest, non-significant increase (negative values, Fig. 7L). This effect was different from CACO-2 cells under K8 knockdown (Fig. 4) and might indicate some degree of cellular stress in the epithelium in some animals. Conversely, pT555 signal was greatly and significantly decreased in all knockout animals as expected (Fig. 7K,L, where n=3 and n=4, respectively). Because the best-known function of atypical PKC is to maintain the structure and localization of tight junctions (Hurd et al., 2003) and paracellular permeability (Hirose et al., 2002), we hypothesized that a decrease in pT555 PKC might affect tight junctions in the upper part of villi. We did not observe any morphological changes by electron microscopy (not shown). However, transepithelial uptake of fluorescent Dextran 3000, a bona fide probe for paracellular permeability, was significantly increased in small intestine loops with their normal blood supply (Fig. 7M). No evidence of inflammation was observed microscopically, or by myeloperoxidase assay of the intestinal mucosa in the same animals (control, 0.124±0.05 OD units/50 µg protein; K8-null, 0.05±0.08 OD units/50 µg protein). Therefore, the decrease in PKC correlates with a modest but significant increase in permeability of the tight junction in the small intestine, as predicted by previous studies on aPKC functions.
Hsp70 and filamentous keratins are essential for the rephosphorylation of the turn motif in mature PKC
The results described above indicate that Hsp70 and the keratin cytoskeleton are necessary to maintain the steady-state levels of PKC but do not distinguish between direct participation of those molecules in the rescue mechanism and indirect effects mediated by other molecules, such as kinases. To independently corroborate the role of Hsp70 and the cytoskeleton in the maintenance of PKC levels we designed an in-vitro rescue of PKC turn motif phosphorylation assay, using different cellular fractions. Because PKC lacks the phorbol-ester binding domain, it was impossible to dephosphorylate PKC by phorbol-ester overstimulation, as with conventional PKCs. Therefore, we incubated S1, S2 and P fractions of CACO-2 cells (described in Figs 1 and 2) in the presence of ATP and an excess of the consensus PKC substrate peptide. The experiments were performed in the presence of proteases and proteasome inhibitor cocktails, but in the absence of phosphatase inhibitors.
After 5-hour incubations with the PKC substrate peptide, we succeeded in substantially decreasing the pT555 PKC signal in all three fractions, without noticeable changes in the PKC protein levels (Fig. 8A for S1, not shown for S2 and P; the fractions containing dephosphorylated PKC are hereafter referred to as S1* and P*). Then, the peptide was removed by ultrafiltration, and ATP was replenished. Separately, none of the fractions were able to rephosphorylate PKC (S1*+ATP, Fig. 8A; P*+ATP, Fig. 8B). Mixing S2 with either S1 or P in the presence of ATP also failed to achieve any PKC rephosphorylation (not shown). However, when fractions S1* and P* were mixed together in the presence of ATP, pT555 signal was restored, in most cases to levels similar to those at the beginning of the experiment (S1*+P*+ATP, Fig. 8C). Because the P fraction is enriched in keratins but still contains keratin-associated proteins, we also used a highly purified preparation of keratins to supplement S1*. When analyzed by two-dimensional gels and silver stain, these keratins were found to be >99.9% pure [no contaminants were detected within the sensitivity of silver stain; supplementary material Fig. S2A (Oakley et al., 1980)]. Such a preparation was found to be as equally active as the P fraction in supplementing S1* for PKC rephosphorylation (S1*+IF+ATP, Fig. 8D). By contrast, single isolated non-filamentous K8, K18, or K19 failed as supplements for S1* to induce PKC rephosphorylation (Fig. 8E). The specificity of Hsp70 and/or Hsc70 for the PKC rephosphorylation was tested by immunodepleting S1* with three anti-Hsp70 and anti-Hsc70 antibodies. As expected, after immunodepletion of Hsp70 and/or Hsc70, S1* supplemented with purified filamentous keratins also failed to rephosphorylate PKC (Hsp70-depleted, S1*+IF+ATP, Fig. 8F). However, if the same immunodepleted S1* fraction was supplemented with recombinant purified Hsp70 (single-band by silver stain, supplementary material Fig. S2B), it regained its ability to rephosphorylate pT555 PKC (Fig. 8G).
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Finally, we wanted to check whether Hsp70 that is bound to IF in the pellet fraction (P) of CACO-2 cells is functionally competent to enable PKC rephosphorylation. To that end, we added the intermediate filament pellet P* fraction to the PKC-dephosphorylated Hsp70-immunodepleted S1* fraction (Fig. 8H). Indeed, we found that addition of the P* fraction and ATP (+P+ATP, Fig. 8H) resulted in PKC rephosphorylation, whereas purified IFs, lacking Hsp70, did not rescue pT555 signal (+IF+ATP, Fig. 8H) in the presence of Hsp70-immunodepleted S1* (Fig. 8H). We conclude that Hsp70 and/or Hsc70 and filamentous keratins, present together in an insoluble cytoskeletal pellet compatible with the complexes shown in vitro in Fig. 3, are equally necessary to rescue dephosphorylated PKC in the inactive conformation and enable rephosphorylation of the turn motif.
Overexpression of keratin filaments affects the subcellular distribution of PKC
Bearing in mind that there is an excess of soluble Hsp70 in intestinal cells (Fig. 1), and that filamentous keratins are essential for the rescue of PKC phosphorylation (Fig. 8), we hypothesized that overexpression of keratin filaments might affect the distribution of PKC within an epithelial cell, because overexpressed keratins result in redistributed and mislocalized excessive IFs (Wald et al., 2005). The rationale was that the assembly of the ternary complex (PKC, Hsp70, filamentous keratins) might be the rate-liming step for PKC rescue, considering that the keratin-Hsp70 compartment is much smaller than the cytosolic compartment of soluble Hsp70 (Table 1). The prediction was that additional IFs would increase the ability of the cell to rescue more PKC. Furthermore, the additional newly refolded and rephosphorylated PKC would be expected to interact with nearby membranes through its C-terminal domain and become mislocalized. To test these predictions of the model, mild K8 overexpressors (approximately twofold increase in K8 protein) were used first. Unlike `severe' HK8-4 animals with an intestinal phenotype (approximately fourfold increase in K8) (Casanova et al., 1999; Wald et al., 2005), these animals display only a chronic pancreatic phenotype (Toivola et al., 2008). The typical distributions of pT555 PKC (Fig. 9B, arrows) and Hsc70 (Fig. 9J) were not changed by K8 overexpression, but, for both of them, a signal above background was visualized in the cytoplasm, especially in regions where abundant supernumerary IFs were observed (arrows, Fig. 9E-F; Fig. 9M-N). Second, we also analyzed the distribution of Hsc70 and pT555 PKC in villous enterocytes of an independent cohort of five hemizygous HK8-4 animals (`severe' overexpressors). A result identical to the mild overexpressors was observed in flurorescence (not shown). SDS extracts from purified enterocytes of the HK8-4 animals showed the expected increase in keratin, and also a significant fivefold increase in pT555 signal (Fig. 9Q,R). We conclude that IF overexpression does not interfere with the normal localization mechanisms of PKC through its PAR partners, but does increase steady-state levels of phosphorylated aPKC turn motif, possibly through an increased rescue, and results in delocalization of pT555 PKC signal.
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is causative of neoplasia, understanding PKC rescue might be important for interventions in abnormal cell growth. From a functional perspective, the apical polarity phenotype of keratin knockdown (Salas et al., 1997) or knockout (Ameen et al., 2001; Toivola et al., 2004; Satoh et al., 1999) has been documented, and includes loss or mistargeting of apical membrane proteins, depolarization of 
-tubulin, and disorganization of microtubules. It is conceivable that some of these anomalies might be due to the steep decrease in active PKC activity. In agreement with this notion, here we report an increase in paracellular permeability in the intestine (Fig. 7M), as expected from the functions of aPKC on tight junction assembly and integrity reported by others (Hirose et al., 2002).
The data in this work, along with previous publications (Satohisa et al., 2005), support a model of two different apical PKC pools, one associated with PAR3 at the tight-junction, and the other located in the apical membrane, away from junctions (Fig. 10A). In vivo, the latter is observed in mouse intestinal crypts, which CACO-2 cells mimic well, whereas the former is observed in the more differentiated villus enterocytes. Upon continuous activity, PKC from both pools becomes dephosphorylated and acquires an inactive conformation (Fig. 10B). This continuous and rapid turnover was documented here by blocking proteasomal activity (supplementary material Fig. S3). Part of the inactive PKC is ubiquitinylated and degraded (Fig. 10C), but a fraction is rescued by Hsc70 in association with keratins (Fig. 10E), to be rephosphorylated and relocalized back to its normal apical compartments. We do not know what determines the fate of an inactive PKC molecule, although the data in Fig. 3 encourage the speculation that binding to IFs might be the first step of the rescue process. The pull-down data suggest that there are at least two subpopulations of PKC in the inactive conformation bound to IFs: one bound directly and another one attached via a ternary complex that includes Hsp70 and/or Hsc70. We cannot dissect the role of each population, but the functional data (Fig. 8) strongly suggest that the latter represents a PKC in the process of being refolded.
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To further test the notion that the ternary complex of inactive PKC, Hsp70 and IFs might represent the first step of the rescue process, we verified that Hsp70 and/or Hsc70 bound to IFs (Fig. 1D) are functional, because the P* fraction is sufficient to rescue Hsp70-immunodepleted S1 activity to rephosphorylate the turn motif (Fig. 8H). However, the existence of pT555 and active PKC in the IF pellet (Figs 1, 2) suggests that the release step might occur after rephosphorylation. In the absence of a positive identification of the kinase responsible for rephosphorylating the turn motif, we are currently unable to analyze the steps that follow the interaction of PKC with Hsp 70 and/or Hsc70. However, it was surprising that PKC in the IF pellet can be functional in the absence of all its known PAR partners and membranes, just associated with the keratin cytoskeleton. Whereas the size of PKC cytoskeletal compartment is modest (Table 1), the presence of an active PKC pool on the cytoskeleton, away from the plasma membrane, might have important implications for other functions (e.g. membrane traffic) involving PKC-dependent phosphorylations within 1 µm or less from the plasma membrane.
The reconstitution experiments enabled us to independently conclude that there is a direct role for Hsp70 and filamentous keratin in PKC conformational rescue behind the phenotype of keratin knockdown and knockouts. However, there remains the possibility that the keratin knockdown effect might be mediated by the observed decrease in steady-state Hsp70 levels. This possibility seems to be dispelled by the high levels of Hsc70 in K8-null enterocytes. Conversely, the Hsp70 knockdown did not affect the steady-state amount of keratins. Together, these data allow us to conclude that filamentous keratins and Hsp70 are independent but concurrent requirements for the first step of the PKC rescue mechanism, acting in a complex attached to the cytoskeleton.
The apical distribution of keratin IF in intestinal cells is conserved from worms to vertebrates (Oriolo et al., 2007a). From the results in Fig. 8, it seems safe to conclude that the rescue of PKC would occur only at locations where IFs and Hsp70 are present together. Because there is an excess of soluble Hsp70, the availability of IFs in the cell is expected to be a rate-limiting step in the PKC rescue mechanism. We tested this prediction of the model in two different K8-overexpression animal models. In both cases, there was grossly mislocalized pT555 PKC signal in the regions of the cell where abnormal, excessive IFs were observed. Yet, the typical localization to the tight junctions was not affected (Fig. 9), indicating that the canonical localization mechanisms were not disturbed by keratin overexpression.
In summary, we conclude that in the intestinal epithelium IFs display a non-mechanical function, compartmentalizing the Hsp70-dependent rescue of PKC. Results in Table 2 and Table 3 suggest that a similar mechanism might apply to other members of the ABC kinase family with the notable exception of PKC. Interestingly, Akt1, a key signaling molecule for cell survival, might be within the set of Hsp70 and/or Hsc70 clients that depend on filamentous keratins (Tables 2,3). This unsuspected possibility might be important to understand how keratin knockouts sensitize cells for apoptosis. Conversely, the dependence of IF for kinase rescue is clearly not universal for all Hsp70 clients. Such a compartmentalization provides an additional, novel mechanism for sustaining the normal steady-state levels of active atypical PKC along with its functions, also aiding to preserve its normal asymmetric distribution.
The data presented here adds to a growing body of evidence indicating that IFs are integral parts of signaling networks (Pallari and Eriksson, 2006; Kim and Coulombe, 2007). This might be especially important to at the time of understanding phenotypes resulting from IF protein mutations. Such phenotypes might not be the consequence of `mechanical' weakness of the filaments, but rather emerging from dysfunction of signaling pathways. It will be important to incorporate this notion into the testable hypotheses of future research efforts in this field.
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(BD Biosciences); rabbit anti-PKC (Santa Cruz Biotechnology); rabbit anti-phospho-T555 (pT555) PKC (Biosource Invitrogen); rabbit anti-phospho turn motif aPKC (Epitomics); PKC (atypical) pT410 and PKB (Akt) pT308 (Cell Signaling); mouse anti-V5 (Invitrogen); mouse anti-K8 (Biomeda); anti-pan-cytokeratin (Sigma); anti-K8 TROMA I (Hybridoma Bank); rabbit anti-Hsc70 (constitutive form of Hsp70; Stressgen Bioreagents); rabbit anti-Hsp70 (inducible form of Hsp70; Stressgen Bioreagents); rabbit anti-Hsp70 (against constitutive and inducible forms of Hsp70; Cell Signaling), rabbit polyclonal anti-caspase 3 (detects full length caspase-3 and its large cleavage fragment; Cell Signaling); mouse anti-actin (C4MP; Biomedicals); rabbit anti-PAR6 (Abcam); rabbit anti-PAR3 (Upstate); mouse anti-pan-14.3.3 (clone CG15; Lab Vision); rabbit anti-Pals1 (Upstate) and mouse anti-ZO1 (Zymed Laboratories). Affinity-purified secondary antibodies with no cross-reactivity were obtained from Jackson ImmunoResearch Laboratories. Recombinant purified K8, K18 and K19 were obtained from US Biological. Recombinant active Hsp70 was obtained from Stressgen. Recombinant PKC was from Invitrogen. Protease inhibitor cocktail (Sigma, cat. no. P-8340) two phosphatase inhibitor cocktails (Calbiochem, cat. no. 524624 and 52625), and proteasome inhibitor MG-132 (Calbiochem, cat. no. 474790) were used.
Cell cultures, lentivirus production and infection
CACO-2 cells were obtained from American Type Culture Collection. The cells were cultured as described (Salas, 1999). HEK 293TN cells were obtained from System Biosciences as a part of the lentivirus packaging system. PKC shRNA (5'-CCGGGCCTGGATACAATTAACCATTCTCGAGAATGGTTAATTGTATCCAGGCTTTTT-3') was obtained from Open Biosystems (cat. no. TRCN0000006037) in the pLKO.1 lentivirus vector. Anti-Hsc70 shRNA (5'-AGGCCTTTCCAAGATTGCTGTTTAGTGAAGCCACAGATGTAAACAGCAATCTTGGAAAGGCCC-3') in the pLKO.1 lentivirus vector (cat. no. TRCN000017279) and anti-Hsp70 shRNA (5'-AGGCCTTTCCAAGATTGCTGTTTAGTGAAGCCACAGATGTAAACAGCAATCTTGGAAAGGCCC-3') in pGIPZ vector (cat. no. V2LHS_243542) were from Open Biosystems. Mission shRNA lentiviral particles carrying shRNA against human K8 (NM_002273) were from Sigma (5'-CCGGGCAGCTATATGAAGAGGAGATCTCGAGATCTCCTCTTCATATAGCTGCTTTTTG-3', cat. no. SHVRSC-TRCN0000062386 and 5'-CCGGGCCTCCTTCATAGACAAGGTACTCGAGTACCTTGTCTATGAAGGAGGCTTTTTG-3', cat. no. SHVRSC-TRCN0000062384). Lentiviral packaging of the vector was done as described earlier (Wald et al., 2007). CACO-2 cells were typically infected at 2-4 days after seeding and selected with puromycin (5 µg/ml) for 4-12 days.
CACO-2 cell extraction and fractionation
The procedure was modified from Steinert et al. (Steinert et al., 1982). At 10 days after seeding, cells were washed in PBS and then extracted in PBS containing 1% Triton X-100, 2 mM EDTA (extraction buffer, EB) supplemented with cocktails of protease and phosphatase inhibitors (Calbiochem) at room temperature. After 15 seconds (three 5-second intervals) of sonication the cell extract was spun for 10 minutes at 16,000 g). This first supernatant was referred to as the S1 fraction. The pellet was resuspended in 1.5 M KCl, sonicated for 15 seconds (three intervals), incubated for 10 minutes on ice, and spun for 10 minutes at 16,000 g). The resulting supernatant was referred to as the S2 fraction, and the pellet was referred to as the P fraction. For functional assays, S1 was used directly, S2 desalted by ultrafiltration, and P resuspended as a coarse suspension by a 3-second sonication. For immunoblots, S1 and S2 were acetone-precipitated, washed in double-distilled H2O, and resuspended in sample buffer. In all cases protein was determined by Lowry assay.
PKC activity assay
PKC activity was measured using a PKC Kinase Non-Radioactive Assay kit (Assay Designs, Stressgen, Ann Arbor, MI) as follows: CACO-2 cells were grown to confluency for 10 days in 75-mm plates, serum starved for 24 hours, washed twice with PBS and subjected to three fractions preparation (S1, S2 and P) as described above. About 18-20 µg of protein was diluted into the kinase assay dilution buffer (Stressgen) and loaded per well in 96-well plates coated with a PKC substrate peptide. Inhibitors were added to appropriate wells in the following concentrations: 1 µM PKA inhibitor H-89, 1 µM GF10923X (at this concentration inhibiting only conventional and novel PKCs), 1 µM Ro-31-8220 (inhibits atypical PKCs more effectively). The PKC assay was performed according to manufacturer's specifications.
Non-denaturing PKC purification, blot overlay, pull-down assays and immunoprecipitation
PKC was transfected into HEK293TN cells, harvested, purified on Ni2+ columns under non-denaturing conditions and used in blot overlays as described (Wald et al., 2008). Pull-down experiments were performed as described elsewhere (Oriolo et al., 2007b). Briefly, highly purified intermediate filaments from CACO-2 cells were covalently bound to CNBr-activated Sepharose beads (Amersham) according to manufacturer's protocol (25 µg of keratin/10 mg beads per reaction). The beads were blocked with 1% casein, incubated with 2 µg/ml recombinant PKC (Invitrogen) in the presence (2 µg/ml) or absence of recombinant Hsp70 (Stressgen) in PBS containing 1% Triton X-100 overnight at 4°C with gentle shaking and then extensively washed. Sepharose beads coupled to normal rabbit serum (Jackson ImmunoResearch Laboratories) served as a negative control. Immunoprecipitation of PKC was performed as described before (Wald et al., 2005), except that the protein-A-coupled beads (Santa Cruz Biotechnology) were used instead of Sepharose beads. A rabbit polyclonal anti-PKC antibody (Santa Cruz Biotechnology) was used for immunoprecipitation.
Transgenic mice, analyses of intestinal epithelium, and immunofluorescence
The K8-null transgenic mice in the FVB/n background were originally obtained by Baribault and coworkers (Baribault et al., 1994), and the intestinal phenotype is described elsewhere (Ameen et al., 2001). Mice deficient in intermediate filaments in intestinal crypts [K18+/–, K19–/–, hK18 R89C (K18R89C animals) (in a mixed FVB/n x C57BL/6 x 129P2 genetic background)] and K8 overexpressors have been described (Hesse et al., 2007; Toivola et al., 2008; Casanova et al., 1999). Isolation of villus enterocytes was described by McNicholas et al. (McNicholas et al., 1994). For paracellular permeability assays, animals were deeply anesthetized, and a 10-cm loop comprising duodenum and the first part of the jejunum was cut, leaving the normal blood supply. A solution of 10 mM Texas-red-coupled Dextran 3000 (Invitrogen) in 1:1 H2O:PBS supplemented with 5 mg/ml glucose was instilled into the lumen and then the open ends of the loop were clamped. Blood was collected at time 0 from the tail, and by cardiac puncture at 50 minutes at the time of euthanizing the animal. Texas-red fluorescence was analyzed in 70-µl samples of serum in a spectrofluorometer. Myeloperoxidase assay was performed on mucosa scraped from 2 cm of jejunum just distal to the Dextran-perfused loop, as described elsewhere (Bradley et al., 1982)
Procedures for immunofluorescence, frozen sectioning, and confocal microscopy have been described before (Ameen et al., 2001). Triton X-100 extraction before fixation is described elsewhere (Salas et al., 1988) and was performed with the following modification in the extraction buffer: PBS was supplemented with 0.5% Triton X-100, 1 mM MgCl2, 1 mM EGTA and the Sigma cocktail of antiproteases described above. Trichloroacetic acid (TCA) fixations were performed in 10% TCA in water for 10 minutes. Confocal microscopy was performed in a Leica TCS SP5 confocal microscope. Confocal stacks were normally collected at 0.4-µm intervals under a 63x immersion objective. For XZ three-dimensional reconstructions, the entire stack was cropped in 7-voxel thick volumes, reconstructed, and rotated 90° using SlideBook 4.2 software (Intelligent Imaging Innovations).
Analysis of intermediate filament-dependent rephosphorylation of PKC in the soluble fraction of CACO-2 cells
CACO-2 cells were grown and fractionated (S1, S2 and P) as described above, with the exception that the extraction buffer was not supplemented with phosphatase inhibitors. To induce the activity-dependent dephosphorylation of PKC, the S1, S2 and P fractions were incubated in the presence of 150 µM PKC-substrate peptide (Upstate, cat. no. 12-536) and 1 mM ATP at 30°C with gentle shaking for 5 hours. After treatment, the peptide was removed by ultrafiltration in Centricon YM-3 (Millipore). To assess PKC rephosphorylation, 50 µg of S1 fraction protein was then incubated with 20 µg of the P fraction protein or with 15 µg of purified IFs from CACO-2 cells [obtained as described by Steinert et al. (Steinert et al., 1982)] in the presence or absence of 1 mM ATP at 30°C for 4 hours. For isolated, non-filamentous recombinant keratins, 5 µg of K8, K18 or K19 were used instead of native IFs under the same conditions. Following incubation, the phosphorylation state of PKC was examined by western blot using anti-pT555-PKC antibodies.
To immunodeplete Hsc70 and/or Hsp70 protein, the S1 fraction was incubated with rabbit anti-Hsc70 and two different rabbit anti-Hsp70 antibodies together at 4°C overnight (non-immune rabbit serum served as a control). Then, protein-A beads (Santa Cruz Biotechnology) were added and incubated for 2 hours at 4°C, spun, and the supernatant used in subsequent in vitro PKC rephosphorylation experiments.
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We are deeply thankful to Yolanda Figueroa-Menendez for the excellent technical assistance, to Nevis Fregien for help with lentivirus preparations, and to Richard Rotundo for critically reviewing the manuscript. Supported by NIDDK (NIH) grants RO1DK057805 and R01DK076652 (to P.J.S.) and RO1DK47918 (to M.B.O.). The TROMA I monoclonal antibody developed by Philippe Brulet and Rolf Kemler was obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Department of Biological Sciences. Andrea S. Oriolo was a recipient of a scholarship from Department of Defense training grant 4-49497-LS-HSI, and Flavia A. Wald was a recipient of a Crohn's and Colitis Foundation of America post-doctoral award. Deposited in PMC for release after 12 months.
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Aghdassi, A., Phillips, P., Dudeja, V., Dhaulakhandi, D., Sharif, R., Dawra, R., Lerch, M. M. and Saluja, A. (2007). Heat shock protein 70 increases tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma. Cancer Res. 67, 616-625.
Ameen, N. A., Figueroa, Y. and Salas, P. J. (2001). Anomalous apical plasma membrane phenotype in CK8-deficient mice indicates a novel role for intermediate filaments in the polarization of simple epithelia. J. Cell Sci. 114, 563-575.[Abstract]
Arlander, S. J., Felts, S. J., Wagner, J. M., Stensgard, B., Toft, D. O. and Karnitz, L. M. (2006). Chaperoning checkpoint kinase 1 (Chk1), an Hsp90 client, with purified chaperones. J. Biol. Chem. 281, 2989-2998.
Baribault, H., Penner, J., Iozzo, R. V. and Wilson-Heiner, M. (1994). Colorectal hyperplasia and inflammation in keratin 8-deficient FVB/N mice. Genes Dev. 8, 2964-2973.
Baumler, M. D., Nelson, D. W., Ney, D. M. and Groblewski, G. E. (2007). Loss of exocrine pancreatic stimulation during parenteral feeding suppresses digestive enzyme expression and induces Hsp70 expression. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G857-G866.
Bradley, P. P., Priebat, D. A., Christensen, R. D. and Rothstein, G. (1982). Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J. Invest. Dermatol. 78, 206-209.[CrossRef][Medline]
Broquet, A. H., Thomas, G., Masliah, J., Trugnan, G. and Bachelet, M. (2003). Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release. J. Biol. Chem. 278, 21601-21606.
Casanova, M. L., Bravo, M., Ramirez, A., Morreale de Escobar, G., Were, F., Merlino, G., Vidal, M. and Jorcano, J. L. (1999). Exocrine pancreatic disorders in transgenic mice expressing human keratin 8. J. Clin. Invest. 103, 1587-1595.[Medline]
Chen, D., Gould, C., Garza, R., Gao, T., Hampton, R. Y. and Newton, A. C. (2007). Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. J. Biol. Chem. 282, 33776-33787.
Daugaard, M., Rohde, M. and Jäättelä, M. (2007). The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Lett. 581, 3702-3710.[CrossRef][Medline]
Doong, H., Rizzo, K., Fang, S., Kulpa, V., Weissman, A. M. and Kohn, E. C. (2003). CAIR-1/BAG-3 abrogates heat shock protein-70 chaperone complex-mediated protein degradation: accumulation of poly-ubiquitinated Hsp90 client proteins. J. Biol. Chem. 278, 28490-28500.
Dutil, E. M., Keranen, L. M., DePaoli-Roach, A. A. and Newton, A. C. (1994). In vivo regulation of protein kinase C by trans-phosphorylation followed by autophosphorylation. J. Biol. Chem. 269, 29362-29359.
Fields, A. P. and Regala, R. P. (2007). Protein kinase C iota: human oncogene, prognostic marker and therapeutic target. Pharmacol. Res. 55, 487-497.[CrossRef][Medline]
Freeman, B. C. and Morimoto, R. I. (1996). The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding. EMBO J. 15, 2969-2979.[Medline]
Gao, T. and Newton, A. C. (2002). The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C. J. Biol. Chem. 277, 31585-31592.
Gao, T. and Newton, A. C. (2006). Invariant Leu preceding turn motif phosphorylation site controls the interaction of protein kinase C with Hsp70. J. Biol. Chem. 281, 32461-32468.
Gao, T., Brognard, J. and Newton, A. C. (2008). The phosphatase PHLPP controls the cellular levels of Protein Kinase C. J. Biol. Chem. 283, 6300-6311.
Haag Breese, E., Uversky, V. N., Georgiadis, M. M. and Harrington, M. A. (2006). The disordered amino-terminus of SIMPL interacts with members of the 70-kDa heat-shock protein family. DNA Cell Biol. 25, 704-714.[CrossRef][Medline]
Hansra, G., Garcia-Paramio, P., Prevostel, C., Whelan, R. D., Bornancin, F. and Parker, P. J. (1999). Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes. Biochem. J. 342, 344-337.
Hesse, M., Grund, C., Herrmann, H., Bröhl, D., Franz, T., Omary, M. B. and Magin, T. M. (2007). Mutation of keratin 18 within the coil 1A consensus motif causes widespread keratin aggregation but cell type-restricted lethality in mice. Exp. Cell Res. 313, 3127-3140.[CrossRef][Medline]
Hirasaka, K., Tokuoka, K., Nakao, R., Yamada, C., Oarada, M., Imagawa, T., Ishidoh, K., Okumura, Y., Kishi K. and Nikawa, T. (2008). Cathepsin C propeptide interacts with intestinal alkaline phosphatase and heat shock cognate protein 70 in human Caco-2 cells. J. Physiol. Sci. 58, 105-111.[CrossRef][Medline]
Hirose, T., Izumi, Y., Nagashima, Y., Tamai-Nagai, Y., Kurihara, H., Sakai, T., Suzuki, Y., Yamanaka, T., Suzuki, A., Mizuno, K. et al. (2002). Involvement of ASIP/Par-3 in the promotion of epithelial tight junction formation. J. Cell Sci. 115, 2485-2495.
Hurd, T. W., Gao, L., Roh, M. H., Macara, I. G. and Margolis, B. (2003). Direct interaction of two polarity complexes implicated in epithelial tight junction assembly. Nat. Cell Biol. 5, 137-142.[CrossRef][Medline]
Kim, S. and Coulombe, P. A. (2007). Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm. Genes Dev. 21, 1581-1597.
Lang, W., Wang, H., Ding, L. and Xiao, L. (2004). Cooperation between PKC-alpha and PKC-epsilon in the regulation of JNK activation in human lung cancer cells. Cell. Signal. 16, 457-467.[CrossRef][Medline]
Liao, J., Lowthert, L. A., Ghori, N. and Omary, M. B. (1995). The 70-kDa heat shock proteins associate with glandular intermediate filaments in an ATP-dependent manner. J. Biol. Chem. 270, 915-922.
Liu, T. S., Musch, M. S., Sugi, K., Walsh-Reitz, M. M., Ropeleski, M. J., Hendrickson, B. A., Pothoulakis, C., Lamont, J. T. and Chang, E. B. (2003). Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction. Am. J. Physiol. Cell Physiol. 284, C1073-C1082.
Magin, T. M., Vijayaraj, P. and Leube, R. E. (2007). Structural and regulatory functions of keratins. Exp. Cell Res. 313, 2021-2032.[CrossRef][Medline]
Marceau, N., Schutte, B., Gilbert, S., Loranger, A., Henfling, M. E. R., Broers, J. L. V., Mathew, J. and Ramaekers, F. C. S. (2007). Dual roles of intermediate filaments in apoptosis. Exp. Cell Res. 313, 2265-2281.[CrossRef][Medline]
McNicholas, C. M., Brown, C. D. A. and Turnberg, L. A. (1994). Na-K-Cl cotransport in villus and crypt cells from rat duodenum. Am. J. Physiol. 267, G1004-G1011.[Medline]
Newton, A. C. (2003). Regulation of the ABC kinases by phosphorylation: PKC as a paradigm. Biochem. J. 370, 361-371.[CrossRef][Medline]
Nollen, E. A. and Morimoto, R. I. (2002). Chaperoning signaling pathways: molecular chaperones as stress-sensing `heat shock' proteins. J. Cell Sci. 115, 2809-2816.
Oakley, B. R., Kirsch, D. R. and Moris, N. R. (1980). A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105, 361-363.[CrossRef][Medline]
Omary, M. B., Coulombe, P. A. and McLean, W. H. (2004). Intermediate filament proteins and their associated diseases. New Engl. J. Med. 351, 2087-2100.
Oriolo, A. S., Wald, F. A., Ramsauer, V. P. and Salas, P. J. I. (2007a). Intermediate filaments: a role in epithelial polarity. Exp. Cell Res. 313, 2255-2264.[CrossRef][Medline]
Oriolo, A. S., Wald, F. A., Canessa, G. and Salas, P. J. I. (2007b). GCP6 binds to intermediate filaments: a novel function of keratins in the organization of microtubules in epithelial cells. Mol. Biol. Cell 18, 781-794.
Pallari, H. M. and Eriksson, J. E. (2006). Intermediate filaments as signaling platforms. Sci STKE 2006, pe53.
Pang, H., Le P. U. and Nabi, I. R. (2004). Ganglioside GM1 levels are a determinant of the extent of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus. J. Cell Sci. 117, 1421-1430.
Pierzchalski, P., Krawiec, A., Gawelko, J., Pawlik, W. W., Konturek, S. J. and Gonciarz, M. (2008). Molecular mechanism of protection against chemically and gamma-radiation induced apoptosis in human colon cancer cells. Physiol. Pharmacol. 59 Suppl. 2, 191-202.
Pinto, M., Robine-Leon, S., Appay, M. D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J. et al. (1983). Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47, 323-330.
Planko, L., Böhse, K., Höhfeld, J., Betz, R. C., Hanneken, S., Eigelshoven, S., Kruse, R., Nöthen, M. M. and Magin, T. M. (2007). Identification of a keratin-associated protein with a putative role in vesicle transport. Eur. J. Cell Biol. 86, 827-839.[CrossRef][Medline]
Rose, L. S. and Kemphues, K. J. (1998). Early patterning of the C. elegans embryo. Annu. Rev. Genet. 32, 521-545.[CrossRef][Medline]
Salas, P. J. (1999). Insoluble gamma-tubulin-containing structures are anchored to the apical network of intermediate filaments in polarized CACO-2 epithelial cells. J. Cell Biol. 146, 645-658.
Salas, P. J., Vega-Salas de Hochman, J., Rodriguez-Boulan, E. and Edidin, M. (1988). Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity. J. Cell Biol. 107, 2363-2376.
Salas, P. J., Rodriguez, M. L., Viciana, A. and Vega-Salas de Hauri, H. P. (1997). The apical sub-membrane cytoskeleton participates in the organization of the apical pole in epithelial cells. J. Cell Biol. 137, 359-375.
Satoh, M. I., Hovington, H. and Cadrin, M. (1999). Reduction of cytochemical ecto-ATPase activities in keratin 8-deficient FVB/N mouse livers. Med. Electron Microsc. 32, 209-212.[Medline]
Satohisa, S., Chiba, H., Osanai, M., Ohno, S., Kojima, T., Saito, T. and Sawada, N. (2005). Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4a-induced epithelial polarization. Exp. Cell Res. 310, 66-78.[CrossRef][Medline]
Savart, M., Letard, P., Bultel, S. and Ducastaing, A. (1992). Induction of protein kinase C down-regulation by the phorbol ester TPA in a calpain/protein kinase C complex. Int. J. Cancer 52, 399-403.[Medline]
Singh, O. V., Pollard, H. B. and Zeitlin, P. L. (2008). Chemical rescue of deltaF508-CFTR mimics genetic repair in cystic fibrosis bronchial epithelial cells. Mol. Cell Proteomics 7, 1099-1110.
Steinert, P., Zackroff, R., Aynardi-Whitman, M. and Goldman, R. D. (1982). Isolation and characterization of intermediate filaments. Methods Cell Biol. 24, 399-419.[Medline]
Suzuki, A. and Ohno, S. (2006). The PAR-aPKC system: lessons in polarity. J. Cell Sci. 119, 979-987.
Toivola, D. M., Krishnan, S., Binder, H. J., Singh, S. K. and Omary, M. B. (2004). Keratins modulate colonocyte electrolyte transport via protein mistargeting. J. Cell Biol. 164, 911-921.
Toivola, D. M., Tao, G. Z., Habtezion, A., Liao, J. and Omary, M. B. (2005). Cellular integrity plus: organelle-related and protein-targeting functions of intermediate filaments. Trends Cell Biol. 15, 608-617.[CrossRef][Medline]
Toivola, D. M., Nakarnichi, I., Stmad, P., Michie, S. A., Ghori, N., Harada, M., Zeh, K., Oshima, R. G., Baribault, H. and Omary, M. B. (2008). Keratin overexpression levels correlate with the extent of spontaneous pancreatic injury. Am. J. Pathol. 172, 882-892.
van den Ijssel, P., Norman, D. G. and Quinlan, R. A. (1999). Molecular chaperones: small heat shock proteins in the limelight. Curr. Biol. 9, R103-R105.[CrossRef][Medline]
Wald, F. A., Oriolo, A. S., Casanova, M. L. and Salas, P. J. (2005). Intermediate filaments interact with dormant ezrin in intestinal epithelial cells. Mol. Biol. Cell 16, 4096-4107.
Wald, F. A., Oriolo, A. S., Mashukova, A., Fregien, N. L., Langshaw, A. H. and Salas, P. J. I. (2008). Atypical protein kinase C (iota) activates ezrin in the apical domain of intestinal epithelial cells. J. Cell Sci. 121, 644-654.
Zhang, X., Zhu, J., Yang, G. Y., Wang, Q. J., Qian, L., Chen, Y. M., Chen, F., Tao, Y., Hu, H. S., Wang, T. et al. (2007). Dishevelled promotes axon differentiation by regulating atypical protein kinase C. Nat. Cell Biol. 9, 743-754.[CrossRef][Medline]
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