Keratin intermediate filaments are heteropolymers of type I and type II polypeptides that constitute the bulk of the epithelial cytoskeleton. We microinjected seven keratin monoclonal antibodies into human epithelial cells, and two of them, only A45-B/B3 and LP3K, caused the formation of keratin aggregates. The keratin filaments in human epithelial cells were also disrupted by a monovalent A45-B/B3 Fab fragment, suggesting that the binding of the antibody, rather than cross-linking, collapses the filaments. Immunoblotting and ELISA experiments suggested that the antibody reacted weakly with recombinant K8 but did not react with recombinant K18 at all. However, the antibody reactivity increased substantially when a mixture of the two keratin polypeptides, either recombinant or derived from MCF-7, was used. The epitopes of 15 monoclonal antibodies recognizing human K8 were characterized by their reactivity with recombinant fragments of K8. Reactivity of antibody A45-B/B3 with fragments of K8 in the presence of K18 revealed that the antibody recognizes an epitope in the rod domain of K8, between residues 313 and 332, on the amino-terminal side of the stutter in helix 2B, which is involved in heterotypic association. The data suggest that this region of K8 undergoes a conformational change following interaction with the complementary K18 either to expose the epitope or to increase its affinity for the antibody. Taken together, the data highlight the role of this epitope in heterotypic association and in filament stabilization.

Keratins 8 and 18 (K8/18) are major components of the intermediate filaments (IFs) of simple epithelia. We report here the identification of a novel protein termed trichoplein. This protein shows a low degree of sequence similarity to trichohyalin, plectin and myosin heavy chain, and is a K8/18-binding protein. Among interactions between trichoplein and various IF proteins that we tested using two-hybrid methods, trichoplein interacted significantly with K16 and K18, and to some extent with K5, K6a, K8 and K14. In in vitro co-sedimentation assays, trichoplein directly binds to K8/18, but not with vimentin, desmin, actin filaments or microtubules. An antibody raised against trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was immunoprecipitated using this antibody in a complex with K8/18 and immunostaining revealed that trichoplein colocalized with K8/18 filaments in HeLa cells. In polarized Caco-2 cells, trichoplein colocalized not only with K8/18 filaments in the apical region but also with desmoplakin, a constituent of desmosomes. In the absorptive cells of the small intestine, trichoplein colocalized with K8/18 filaments at the apical cortical region, and was also concentrated at desmosomes. Taken together, these results suggest that trichoplein is a keratin-binding protein that may be involved in the organization of the apical network of keratin filaments and desmosomes in simple epithelial cells.

Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps.

Keratin polypeptide 20 (K20) is an intermediate filament protein with preferential expression in epithelia of the stomach, intestine, uterus, and bladder and in Merkel cells of the skin. K20 expression is used as a marker to distinguish metastatic tumor origin, but nothing is known regarding its regulation and function. We studied K20 phosphorylation as a first step toward understanding its physiologic role. K20 phosphorylation occurs preferentially on serine, with a high stoichiometry as compared with keratin polypeptides 18 and 19. Mass spectrometry analysis predicted that either K20 Ser(13) or Ser(14) was a likely phosphorylation site, and Ser(13) was confirmed as the phospho-moiety using mutation and transfection analysis and generation of an anti-K20-phospho-Ser(13) antibody. K20 Ser(13) phosphorylation increases after protein kinase C activation, and Ser(13)-to-Ala mutation interferes with keratin filament reorganization in transfected cells. In physiological contexts, K20 degradation and associated Ser(13) hyperphosphorylation occur during apoptosis, and chemically induced mouse colitis also promotes Ser(13) phosphorylation. Among mouse small intestinal enterocytes, K20 Ser(13) is preferentially phosphorylated in goblet cells and undergoes dramatic hyperphosphorylation after starvation and mucin secretion. Therefore, K20 Ser(13) is a highly dynamic protein kinase C-related phosphorylation site that is induced during apoptosis and tissue injury. K20 Ser(13) phosphorylation also serves as a unique marker of small intestinal goblet cells.

Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

To elucidate the function of keratins 8 and 18 (K8/18), major components of the intermediate filaments of simple epithelia, we searched for K8/18-binding proteins by screening a yeast two-hybrid library. We report here that human Mrj, a DnaJ/Hsp40 family protein, directly binds to K18. Among the interactions between DnaJ/Hsp40 family proteins and various intermediate filament proteins that we tested using two-hybrid methods, Mrj specifically interacted with K18. Immunostaining with anti-Mrj antibody showed that Mrj colocalized with K8/18 filaments in HeLa cells. Mrj was immunoprecipitated not only with K18, but also with the stress-induced and constitutively expressed heat shock protein Hsp/c70. Mrj bound to K18 through its C terminus and interacted with Hsp/c70 via its N terminus, which contains the J domain. Microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, without effects on the organization of actin filaments and microtubules. Taken together, these results suggest that Mrj may play an important role in the regulation of K8/18 filament organization as a K18-specific co-chaperone working together with Hsp/c70.

Proteome-scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein-protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two-hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver-specific, liver-phenotype and liver-disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver-specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

APE1/Ref-1 (hereafter, APE1), a DNA repair enzyme and a transcriptional coactivator, is a vital protein in mammals. Its role in controlling cell growth and the molecular mechanisms that fine-tune its different cellular functions are still not known. By an unbiased proteomic approach, we have identified and characterized several novel APE1 partners which, unexpectedly, include a number of proteins involved in ribosome biogenesis and RNA processing. In particular, a novel interaction between nucleophosmin (NPM1) and APE1 was characterized. We observed that the 33 N-terminal residues of APE1 are required for stable interaction with the NPM1 oligomerization domain. As a consequence of the interaction with NPM1 and RNA, APE1 is localized within the nucleolus and this localization depends on cell cycle and active rRNA transcription. NPM1 stimulates APE1 endonuclease activity on abasic double-stranded DNA (dsDNA) but decreases APE1 endonuclease activity on abasic single-stranded RNA (ssRNA) by masking the N-terminal region of APE1 required for stable RNA binding. In APE1-knocked-down cells, pre-rRNA synthesis and rRNA processing were not affected but inability to remove 8-hydroxyguanine-containing rRNA upon oxidative stress, impaired translation, lower intracellular protein content, and decreased cell growth rate were found. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus affecting gene expression through posttranscriptional mechanisms.

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.