Development of the Yeast Three-Hybrid System
Because keratin IFs are obligate heteropolymers (Fuchs, 1994
; Parry and Steinert, 1999
), it was essential to study the interaction of plakins with heterodimers by using a developed yeast three-hybrid system (Zhang, 2000
) that allowed the concomitant expression of three proteins, one plakin and two complementary keratins. Previous studies have demonstrated that IF proteins are capable of strong interaction in yeast (Leung and Liem, 1996
; Meng et al., 1996
; Schnabel et al., 1998
). Therefore, we replaced the LEU2 selective marker in the plasmid pACT2 by URA3 to generate pACT2-URA and to have three plasmids available for interaction assays in yeast: one to express GAL4-BD fusion protein (pAS2–1) and two for the expression of GAL4-AD fusion protein (pACT2 or pACT2-URA), each encoding a different selection marker, TRP1, LEU2, or URA3, respectively. By introducing two complementary type I and type II keratins fused to GAL4-AD, one in pACT2 and the other in pACT2-URA, that are expected to dimerize properly (Leung and Liem, 1996
; Meng et al., 1996
; Schnabel et al., 1998
), the effect of heterodimerization of keratins on their interaction with the IF-binding domain of plakins fused to GAL4-BD in pAS2–1 can be studied in yeast.
Selective Binding of BP230 with Dimeric Epidermal K5/K14 Requires Both the COOH-Terminal B and C Subdomains
The complete tail of human BP230 (residues 1881–2649) was shown to be codistributed with the epidermal K5/K14 IF network in transfected SCC-13 keratinocytes (Yang et al., 1996
). To define the ability of human BP230 to associate with keratins and vimentin, we first used the yeast three-hybrid system. Initially, two large BP230 constructs, encompassing either the entire globular COOH tail domain (residues 1881–2649) or a shortened version (residues 1946–2649), respectively, produced GAL4-AD fusion proteins, which caused autotransactivation of reporter genes, precluding yeast three-hybrid analysis. By contrast, a BP230 fragment encompassing the B and C subdomains, BP230-BC residues 2077–2648 (), did not cause autotransactivation and specifically interacted with the K5/K14 heterodimer in yeast three-hybrid assays (). This interaction appeared to be weak, because yeast growth was sustained only on plates lacking histidine, not on plates lacking adenine. Indeed, it is known that in the PJ69–4A strain, the ADE2 reporter gene requires more GAL4 activity than the HIS3 reporter gene (Cagney et al., 2000
Figure 2. Survey of the sites of interaction between the COOH-terminal domain of BP230 fused to GAL4-BD and IF proteins fused to GAL4-AD: + and - indicate growth or no growth, respectively, on selective media. The PJ69–4A yeast strain was cotransformed (more ...)
Neither the single keratins K5, K14, K8, and K18 nor the heterodimeric keratin K8/K18 or vimentin, all fused to the GAL4-AD, bound to BP230-BC (). To further map the sequences essential for binding to K5/K14, we tested a series of deletion mutants of BP230. A construct encompassing the B subdomain and the linker, BP230-BL, or the B subdomain, BP230-B, did not interact with any of the IF proteins tested (). In contrast, the C subdomain, BP230-C, did bind to monomeric K5 and K14 but neither to K8 or K18 nor to dimeric K5/K14, K8/K18, or vimentin (see below). Finally, we deleted a motif situated at the extremity of BP230 2641GXXSXYXXS2649 (where X is not defined), which is also conserved in DP and PL (). Removal of these last eight residues of BP230 from previously active deletion mutants completely abrogated their binding to IF proteins, indicating that the COOH extremity of BP230 is crucial for binding ().
The B and C Subdomains of the BP230 Tail Are Essential for Its Selective Coalignment with K5/K14 IFs
To further investigate whether specific sequences of BP230 are responsible for its association with various types of IFs, we generated expression vectors encoding deletion mutants of the BP230 tail GFP-tagged at the NH2
-terminus. These hybrid proteins, like other members of the plakin family, were found to be more stable and easier to analyze than c-myc–tagged deletion mutants (not shown) (Smith and Fuchs, 1998
). To determine whether deletion mutants of BP230 coaligned with different IF networks, we transiently transfected the above constructs into various cell lines, including PA-JEB/β4 keratinocytes, which contain a K5/K14 keratin network, PtK2 cells containing K8/K18 IFs, and COS-7 cells for vimentin IFs.
Immunofluorescence microscopy studies of transfected PA-JEB/β4 keratinocytes demonstrated that BP2301881–2649, encompassing the entire tail, decorated the epidermal K5/K14 IF network (not shown, and ). In up to 10% of the transfected keratinocytes, the epidermal keratin network had partially collapsed. In contrast, when expressed in PtK2 cells, the BP230 tail was not codistributed with the K8/K18 network but rather remained diffusely distributed in the cytoplasm. The same distribution pattern was observed in transfected COS-7 cells, in which no obvious colocalization with vimentin was seen (not shown, and ). The deletion mutant BP230-BC also coaligned with and, rarely, disrupted the K5/K14 networks of transfected PA-JEB/β4 cells (Figures and ). BP230-BC was not codistributed with either K8/K18 or vimentin IFs but rather was found primarily diffusely distributed in the cytoplasm of PtK2 and COS-7 cells, respectively (not shown, and ). The deletion of the ultimate eight residues from the COOH terminus of BP230-BC abrogated the coalignment of the recombinant BP230-BCΔ8 with K5/K14 in transfected PA-JEB/β4 cells (Figures and ). Finally, in all transfected cell lines, BP230-C was present in small cytoplasmic aggregates, which were not coaligned with and did not disrupt the K5/K14, K8/K18, and vimentin IF networks (). When expressed by itself in the various cell lines tested, GFP was found either diffusely distributed over the cytoplasm or in the nucleus without obvious staining of the IF network (not shown).
Figure 3. Summary of the data obtained in cell transfection experiments in PA-JEB/β4, PtK2, and COS-7 cell lines: + and - indicate colocalization or not between GFP-tagged BP230 or DP fusion proteins and the network of IFs consisting of K5/K14 in PAJEB-β4 (more ...)
Figure 4. The codistribution of BP230 with the epidermal keratin network is mediated by sequences contained within the B and C subdomains and the most COOH-terminal extension. Ec-topic expression of the COOH-terminal domain of BP230 fused to GFP in PA-JEB/β4 (more ...)
Consistent with the results in yeast, the transfection studies indicate that (1) the BP230 tail contains sequences conferring binding specificity for the epidermal K5 and K14 keratins and (2) a region of BP230 encompassing the B and C subdomains, including the intervening linker region, as well as the COOH-terminal extremity is essential for its interaction with K5/K14 filaments. Finally, (3) the observation that BP230-C is unable to become colocalized with the K5/K14 IF network correlates with its failure to interact with dimeric K5/K14 in yeast, suggesting that its binding activity with monomeric K5 and K14 is artificial, most likely as a result of the exposure of cryptic binding sites by the N-terminal truncation.
The Interaction of the DP Tail with IFs Depends on the Heterodimerization of Type I and Type II Keratins in Yeast and Is Regulated by Ser 2849
Because previous studies have demonstrated that the DP tail associates with epidermal and nonepidermal keratins (Stappenbeck et al., 1993
; Kouklis et al., 1994
; Meng et al., 1997
), we investigated the ability of two DP constructs containing its entire tail or the B and C regions, DP-AC and DP-BC, respectively, to bind to monomeric or dimeric keratins K5, K14, K8, K18, or vimentin in yeast. Surprisingly, there was no binding with any of the keratins tested, and only DP-BC bound weakly to vimentin (). It has been observed that only DP mutants carrying the amino acid substitution S2849G were coaligned with keratin IF networks in certain cell lines and showed increased binding activity with vimentin and K8/K18 in yeast (Stappenbeck et al., 1994
; Meng et al., 1997
). Therefore, we next assessed the effect of a mutated DP construct in which Ser 2849 was substituted by Gly (S2849G), thereby disrupting a potential protein kinase A consensus phosphorylation site. As shown in , DP-ACS2849G
bound to dimeric K5/K14, K8/K18, and vimentin in yeast, whereas there was no interaction with monomeric K5, K14, K8, or K18.
Figure 5. Yeast three-hybrid analysis of the interaction between various subdomains of the COOH-terminal tail of DP fused to GAL4-BD and IF proteins fused to GAL4-AD: +, +/- and - indicate growth, slow growth, or no growth, respectively, on selective media, (more ...)
Because Western blot analysis of yeast protein extracts showed that the electrophoretic mobility of DP-C was lower than that of DP-CS2849G
, we tested the possibility that phosphorylation of Ser 2849 in the DP tail was responsible for this mobility change. Therefore, the DP-C and DP-CS2849G
recombinant proteins were immunoprecipitated from yeast extracts, subjected to dephosphorylation by calf intestine alkaline phosphatase (CIAP), and immunoblotted. As shown in , phosphatase treatment results in a shift in the electrophoretic mobility of DP-C, making it indistinguishable from that of DP-CS2849G
. Consistent with previous reports that Ser 2849 is phosphorylated in mammalian cells (Stappenbeck et al., 1994
), these findings suggest that Ser 2849 is phosphorylated in yeast as well. In this context, we tested the possibility that phosphorylation of Ser 2849 modulates the folding of DP tail by regulating intramolecular interactions within this region. However, no evidence was found for a direct association between the extremity of the COOH terminus and the B and C subdomains or between the C and B subdomains in yeast (not shown).
Figure 6. Ser 2849 in the DP tail is phosphorylated in yeast. GAL4-recombinant proteins DP-CS2849G (lanes 1 and 2) and DP-C (lanes 3 and 4) were immunoprecipitated from yeast extracts and subjected (lanes 2 and 4) or not (lanes 1 and 3) to dephosphorylation (more ...)
These results (1) imply that the presence of either K5/K14 or K8/K18 in the form of a heterodimer made an interaction of DP with these keratins possible and (2) suggest that phosphorylation of Ser 2849 in yeast negatively affects the ability of DP to interact with keratins. Nevertheless, the binding activity of the DP constructs, in which this Ser residue was replaced by Asp to try mimicking phosphorylated Ser (DP-ACS2849D and DP-BCS2849D), was unaffected (not shown).
Identification of Sequences within the DP Tail Interacting Specifically with Various IF Proteins
Next, we assessed the requirement of specific sequences within the DP tail important for its binding to various types of IFs using yeast three-hybrid analysis. A construct encompassing the C subdomain and the COOH terminus of DP with the S2849G substitution, DP-CS2849G, was able to bind to K5/K14 and K8/K18 but no longer interacted with vimentin (). Deletion of an additional stretch of 51 residues, comprising the GSRS/T repeats (DP-CΔ51), abolished the interaction with K5/K14 and K8/K18. In contrast, deletion of the same 51 residues from the construct DP-BC (DP-BCΔ51) did not prevent binding to any of the dimeric IF proteins, including keratins. However, the interaction appeared to be weaker than that of DP-BC, as inferred from the delayed growth of yeast cells on medium without adenine. This finding suggested that the COOH-terminal extremity of DP contains residues that contribute to IF binding (). Consistent with this idea, we found that a recombinant protein encompassing the last 79 residues of DP, DP-CtS2849G, showed some binding activity with both K5/K14 and K8/K18.
In contrast, a construct encompassing the B subdomain and a large portion of the linker, DP-BL, did not interact with K5 and K14 either as single or heterodimeric proteins but did interact with K8 and heterodimeric K8/K18 and vimentin. To better define which portion of DP-BL is implicated in binding, we tested a construct containing either the linker region of DP, DP-L, or the B subdomain, DP-B. DP-L was sufficient to mediate binding to K8, K8/K18, and vimentin but not to K18 or to K5 and K14 as either single or heterodimeric proteins. By contrast, DP-B was unable to interact with any of the IF proteins tested. These results indicate that (1) a region of DP encompassing the C subdomain is essential but not sufficient for binding to the epidermal K5/K14 keratin pairs, because sequences contained within either the B and the linker region or the 51 COOH-terminal have an impact on the ability of the C subdomain to interact with K5/K14 (see DP-BCΔ51 and DP-CΔ51); (2) the linker region of DP contains recognition sites for simple epithelial keratins and vimentin; and (3) the COOH extremity displays binding activity with both simple and epidermal keratins.
Distinct Regions within the DP Tail Are Required for Its Coalignment with Various IF Networks
We next performed transfection studies using expression vectors encoding fusion proteins encompassing portions of the DP tail linked to GFP at the NH2
-terminus. To ensure the best codistribution potential of these hybrid proteins with various IF networks (Stappenbeck et al., 1994
), we used DP mutants in which Ser 2849 was replaced by Gly. We first tested the behavior in transfected cells of two deletion mutants, which encompassed the entire tail of DP or the tail domain without the A subdomain, DP-ACS2849G
, respectively. When expressed in PA-JEB/β4 keratinocytes, PtK2 cells, and COS-7 cells, these DP mutants were able to decorate epidermal K5/K14 and simple K8/K18 keratins and vimentin IF networks, respectively (Figures and ). In addition, partial disruption and collapse of these networks was observed occasionally, particularly in cells with high DP fusion protein expression levels (not shown). Additional deletion of 51-amino-acid residues from the COOH-terminal, DP-BCΔ51, had no effect on the codistribution of DP with IFs (). The deletion mutant DP-CS2849G
coaligned with epidermal and simple keratin IF networks, although occasionally, it was also distributed in the cytoplasm in cells with high levels of expression (Figures , , and ). This DP mutant protein did not decorate the vimentin network in transfected COS-7 cells but remained diffusely distributed over the cytoplasm (). Notably, the mutant DP-CΔ51 did not become codistributed with any of the IF networks but instead was diffusely distributed over the cytoplasm (Figures , , and ). In contrast, a DP mutant containing the B subdomain and the linker, DP-BL, became colocalized with both the simple keratin and vimentin networks (Figures and ) but not with epidermal keratins (). Finally, a DP-L recombinant protein encompassing the linker alone also exhibited this codistribution potential but was more frequently diffusely distributed in the cytoplasm than the recombinant protein DP-BL and even more than DP-BC, which contain one and two modules, respectively (). In agreement with the results in yeast and in extension to results of previous studies, our findings clearly indicate that (1) a region encompassing the C subdomain and the COOH extremity contains sequences sufficient for the interaction of DP with epidermal and simple keratin filaments; (2) the presence of the B subdomain and the linker region or the COOH-terminal stretch of 51 residues has an important impact on the potential of the C subdomain and COOH-terminal region to become coaligned with keratin networks; and (3) sequences within the linker region confer additional binding activity for the vimentin IF network and are thus essential for such an interaction.
Figure 7. Distinct subdomains affect the coalignment potential of DP tail with the epidermal K5/K14 keratin network. Representatives of double immunofluorescence microscopy analyses of PA-JEB/β4 keratinocytes transiently transfected with cDNAs encoding (more ...)
Figure 8. The ability of the COOH-terminal tail of DP to codistribute with the K8/K18 IF network is affected by sequences contained within either the B subdomain and the linker or the C subdomain and the COOH-terminal extension. PtK2 cells were transiently transfected (more ...)
The DP Tail Preferentially Associates with Polymeric IF Proteins in In Vitro Binding Assays
Because our data suggest that binding of DP and BP230 to epidermal and simple cytokeratins requires that the keratins are in the form of heterodimers, we tested the ability of DP and BP230 tails to associate with monomeric and polymeric IF proteins in in vitro binding assays. K5, K14, K8, and K18 monomers and keratin and vimentin filaments were immobilized on nitrocellulose membranes and overlaid with recombinant DP and BP230 proteins that were in vitro transcribed and translated and used as fluid-phase ligands. In agreement with our yeast two-hybrid results, the recombinant DP-BCS2849G
interacted strongly with filaments of K5/K14, K8/K18, and vimentin but not with K5, K8, and K18 monomers (), whereas weak associations with K14 were observed. Identical results were found using the recombinant protein DP-BC without the Ser substitution, most likely because the in vitro translated product was not phosphorylated (not shown). Furthermore, the recombinant protein DP-C bound to both K5/K14 and K8/K18, although less efficiently than DP-BCS2849G
. Finally, deletion of the last 51 residues from the tail of DP-C, DP-CΔ51, abrogated these interactions. These results provide strong support for the contention that DP associates preferentially with heterodimers of keratin. Attempts to demonstrate an association of the BP230 tail with keratin filaments in overlay binding assays failed (not shown). It is possible that the interaction of BP230 with keratin filaments is too weak to be detected in our in vitro binding assay. Alternatively, the in vitro translated recombinant form of BP230 used may fold incorrectly or lack posttranslational modifications essential for binding to IFs. Such modifications were recently shown to be critical for the association of PL with IF proteins (Janda et al., 2001
Figure 9. Dot-blot overlay assays of recombinant purified monomeric or polymerized IF proteins with radiolabeled fragments of the tail of DP. Left, 35[S]-radiolabeled c-myc–tagged recombinant forms of DP, DP-BCS2849G, DP-CS2849G, or DP-CΔ51 were (more ...)
Evidence That Sequences within the DP Tail Confer Binding Specificities to Distinct IF Proteins
The assumption that distinct subdomains of the plakin tails are responsible for their selective binding to IF proteins was tested further in yeast by generation of chimeric constructs in which distinct regions of BP230 were swapped with the equivalent sequences in DP (). A chimeric construct consisting of the B subdomain of BP230 fused at its carboxyl extremity with the linker of DP, BP(B)–DP(L), had binding activities similar to those of the linker of DP (). Complementary to this, we swapped the linker region of BP230 with the corresponding region of DP in BP230-BC to obtain BP(BC)–DP(L). Despite this swapping, this latter construct was unable to interact with any of the IF proteins tested (). Finally, a chimeric protein consisting of BP230-BC fused at its COOH extremity with the last 51 residues of DP, BP(BC)–DP(Ct) with or without the S2849G substitution, was able to bind to K5/K14. Most importantly, the chimeric construct carrying the S2849G acquired the ability to interact with K8/K18 and weakly with vimentin. Overall, these observations reveal that the linker region and/or the COOH extremity of DP contain sequences critical for the association with K8/K18 and vimentin. Proper folding and exposure of the recognition sites within the linker appear to be affected by the context of the flanking sequences such as the B and/or C subdomains, as inferred from the behavior of BP(BC)–DP(L).
Figure 10. Yeast three-hybrid analysis of the interaction between IF proteins and chimeric proteins BP230 and DP. + and - indicate growth or no growth, respectively, on selective media, tested as described under MATERIALS AND METHODS and in . # indicates (more ...)
The Head and Tail Domains of K5 and K14 Are Not Needed for Their Interaction with BP230 and DP
Finally, because previous studies have provided strong evidence that the head domain of K1 and K5 contains sequences that mediate binding to DP (Kouklis et al., 1994
; Meng et al., 1997
), we studied the effect of the truncation of the head or the tail domain of K5 (K5ΔH and K5ΔT, respectively) and of K14 (K14ΔH and K14ΔT, respectively) on the binding of DP and BP230 to IF proteins. In yeast two-hybrid assays, we first verified that the deletion of the head or tail domains did not impair the ability of K5 and K14 to associate with each other (data not shown). In accordance with previous studies using other combinations of keratin pairs (Schnabel et al., 1998
), we found that the head or tail domains of K5 and K14 are unnecessary for dimerization. We then tested the binding activity of DP-BCS2849G
, and BP230-BC with various combinations of K5 and K14 with or without their head and tail domains (). The results of all experiments were positive. In contrast, DP-CΔ51 did not bind to the various keratin pair combinations (not shown). These findings strongly suggest that the keratin rod domain is sufficient for binding.
Figure 11. Yeast three-hybrid analysis of the interaction between the COOH terminus of either DP or BP230 and K5/K14 keratins, from which the head or tail domains were removed. + and - indicate growth or no growth, respectively, on selective media, tested as (more ...)