Keratins turn over by ubiquitination in a phosphorylation-modulated fashion

Abstract

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.

Figure 1

In vivo ubiquitination of K8 and K18. BHK cells, cotransfected with the indicated keratin(s) and His-Ub, or HT29 cells were treated overnight with DMSO (0.1%) or with an equal volume of ALLN (in DMSO). Total lysates were prepared by solubilizing the cell pellets in sample buffer, and His-ubiquitinated proteins from keratin-transfected BHK cells were purified using a Ni column after cell solubilization in 8-M urea. Total lysates and Ni column–purified samples were separated by SDS-PAGE then stained with Coomassie blue, or were immunoblotted with antibodies to Ub (His), K8, or K18. Asterisks in A highlight bands that likely correspond to ubiquitinated K8 or K18 in ALLN-treated HT29 cells. Asterisks in C represent species that likely correspond to ubiquitinated K8/18 fragments and/or unmodified K8/18 that is part of the keratin tetramer.

Figure 2

Stabilization of transfected keratins by proteasome inhibition and partitioning of ubiquinated keratins with the insoluble pool. (A) BHK cells were transfected with K8 and/or K18. After 36 h, transfected cells were incubated overnight with DMSO or ALLN as in Fig. 1, then labeled with ³⁵S-met (10 min) followed by immediate harvesting or chasing for 4 h. K8, K18 or K8/18 were precipitated with Abs TS1, DC10, and L2A1, respectively. Immunoprecipitates were analyzed by SDS-PAGE then Coomassie staining and fluorography, or were blotted with antibodies to K8 or K18. Due to significant K8 and K18 degradation and their low overall levels when transfected alone, their immunoprecipitates were visualized by blotting rather than Coomassie staining. Asterisks (lane 1) correspond to induced K18 (transfection of K8 likely induces endogenous hamster K18 expression, see also Fig. 4 C) and to K8 and/or K18 degradation products. (B and C) BHK cells were transfected with K8 and/or K18 and with His-Ub followed by isolation of a cytosolic (C), remaining pellet (P) and total cell lysates (T). Ubiquitinated proteins (NiNTA-purified) were also isolated from each of these fractions followed blotting with anti-K8 and anti-K18 Abs.

Figure 3

Keratin phosphorylation in HT29 and transfected BHK cells. (A) HT29 cells were incubated with DMSO or ALLN as in Fig. 1 followed by immunoprecipitation of K8/18 using Ab L2A1. Precipitates were blotted with site-specific phosphokeratin antibodies: K18 pS33 (Ab 8250), K18 pS52 (Ab 3055), K8 pS73 (Ab LJ4), K8 pS431 (Ab 5B3). Note that ALLN treatment generates the previously described hyperphosphorylated form of K8 (due to pS73), termed hK8 (Liao et al. 1997), that is seen as a faint band just above K8 (lane 2) and is recognized by Ab LJ4 (lane 8). (B and C) BHK cells were cotransfected with His-Ub and vector alone (labeled as none), K8 and/or K18. After 2 d, cells were solubilized directly in sample buffer or used to purify Ub-conjugated proteins with a Ni column. The total lysate and the Ni column–purified proteins were blotted with anti-K8 (B) or anti-K18 (C) antibodies. The bands enclosed within the dotted lines (C, blot b) are nonspecific since they are also observed in vector alone–transfected cells. The antibodies used were: M20 (total K8 pool), LJ4 (K8 pS73), 5B3 (K8 pS431), DC10 (total K18 pool), 3055 (K18 pS52), IB4 (K18 pS33), and 4668 (K18 S33, i.e., the nonphosphorylated epitope). Immunoblotting with Ab 8250, which recognizes K18 pS33 also gave a similar result to that in Fig. 3 C, c (not shown).

Figure 4

Accumulation and turnover of ubiquitinated K8 upon mutation of its phosphorylation sites. (A and B) The indicated K8 constructs and His-Ub were transfected into BHK cells. Cells were then divided into two equivalent fractions, one of which was used to prepare a total lysate and the second was used to isolate ubiquitinated proteins for immunoblotting with anti-K8 Ab. The multiple bands above K8 (lanes 3, 8, and 9) represent hyperphosphorylated and/or ubiquitinated K8. (C) BHK cells were transfected with the indicated K8 (lanes 1–6) or K18 (lanes 7–12) constructs, labeled with ³⁵S-met for 10 min (after incubating with ALLN for 6 h), then chased for 60 min (K8) or 40 min (K18) followed by immunoprecipitation as described in Fig. 2. Equal amounts of the K8 and K18 precipitates were loaded as confirmed by blotting with anti-K8 or anti-K18 antibodies. K8 levels (see text) were quantified by densitometric scanning of the radiolabeled K8 bands after fluorography and exposure to x-ray film. Note that transfection with K8 stabilizes the endogenous low levels of BHK hamster K18 (lanes 1–6). The band indicated by an asterisk in lanes 7–12 likely corresponds to hamster K18.