PKD1 interacts with PKD2 through a probable coiled-coil domain.

Autosomal dominant polycystic kidney disease (ADPKD) describes a group of at least three genetically distinct disorders with almost identical clinical features that collectively affects 1:1,000 of the population. Affected individuals typically develop large cystic kidneys and approximately one half develop end-stage renal disease by their seventh decade. It has been suggested that the diseases result from defects in interactive factors involved in a common pathway. The recent discovery of the genes for the two most common forms of ADPKD has provided an opportunity to test this hypothesis. We describe a previously unrecognized coiled-coil domain within the C terminus of the PKD1 gene product, polycystin, and demonstrate that it binds specifically to the C terminus of PKD2. Homotypic interactions involving the C terminus of each are also demonstrated. We show that naturally occurring pathogenic mutations of PKD1 and PKD2 disrupt their associations. We have characterized the structural basis of their heterotypic interactions by deletional and site-specific mutagenesis. Our data suggest that PKD1 and PKD2 associate physically in vivo and may be partners of a common signalling cascade involved in tubular morphogenesis.

Cloning of putative growth regulatory genes from primary human keratinocytes by subtractive hybridization.

In order to isolate genes that might be involved in regulating human keratinocyte (HKc) growth and/or differentiation, we constructed a cDNA library by subtractive hybridization between primary HKc and FaDu head-and-neck squamous cell carcinoma cells. Among the first set of independent cDNAs that we have isolated, ten correspond to known genes, and two represent novel sequences. Nine of the ten known genes are expressed at significantly lower levels in the majority of the SqCC cell lines in comparison with primary HKc. These include cDNAs that encode keratins K5 and K14 which are cytoskeletal proteins normally expressed in lining epithelia, the 14-3-3 protein stratifin/HME-1, lipocortin-II and CaN19 which are calcium-binding proteins that may play a role in HKc differentiation by regulating protein kinase C, plasminogen-activator inhibitor-2 which is a serine-proteinase inhibitor, HBp17 which is a HKc-specific secreted inhibitor of fibroblast growth factors, integrin alpha 3 which plays a role in the anchoring of keratinocytes to basement membrane, and YL-8, a ras-like protein that probably mediates intracellular protein trafficking. In addition, we isolated two cDNAs, LIS-1 which encodes the 45-kDa intracellular subunit of the platelet-activating factor acetyl-hydrolase, and the unknown sequence HFBCB84 which showed reduced expression in only a small number of tumor lines as compared to HKc. Inactivation or loss of any of these proteins may confer a selective advantage onto squamous epithelial cells and contribute to their malignant transformation.

Vectors for a 'double-tagging' assay for protein-protein interactions: localization of the CDK2-binding domain of human p21.

We report the construction of three new vectors which can be used for the 'double-tagging' assay previously reported [Germino et al., Proc. Natl. Acad. Sci. USA 90 (1993) 933-937]. The vectors include two plasmids (pTrc.BCCP and pTrc.EZZ::BCCP) which encode different 'tags' for the capture of a target protein of interest on a filter coated with either avidin or IgG, respectively. The first plasmid (pTrc.BCCP) encodes the C terminus of the biotin carboxylase carrier protein (BCCP) under the control of the Ptac promoter, while the second produces fusions to an IgG-binding domain (EZZ). The gene encoding a protein of interest can be inserted into these plasmids and thereby direct the production of a fusion protein which is biotinylated in vivo and can bind to avidin, or a fusion protein which can bind to IgG. The third is a positive-selection, phase lambda expression vector (lambdaFJG2) which permits the construction of lacZ::cDNA fusion proteins which retain beta-galactosidase activity. The insertion of an active ecoRVR gene between the cloning sites (EcoRI and HindII or NotI) permits the positive selection of inserts. The C-terminal two-thirds of the mouse retinoblastoma-encoding gene (containing the E1A-binding pocket) was cloned into pTrc.BCCP and pTrc.EZZ::BCCP, while the 13S E1a gene was cloned into lambdaFJG2. We show that the interaction between these two proteins can be detected using the 'double-tagging' filter assay, and that this assay has high sensitivity and specificity for detecting this interaction. Finally, we have used these vectors to localize the CDK2-binding domain of the cyclin-dependent kinase inhibitor, p21. These results closely correspond to those obtained using the yeast two-hybrid assay, as well as in vitro binding assays.

Screening for protein-protein interactions.

The assays described above can be used to screen for cellular proteins that can interact with a protein of interest, to screen for mutant proteins that retain the ability to bind to its partner, and to identify the domains and amino acids involved in known protein-protein interactions. Nonetheless, the biological significance of some of these interactions needs to be confirmed by appropriate cellular studies.

Minimally invasive staging of patients with melanoma: sentinel lymphadenectomy and detection of the melanoma-specific proteins MART-1 and tyrosinase by reverse transcriptase polymerase chain reaction.

BACKGROUND: A minimally invasive standard has yet to be developed for sentinel lymphadenectomy, and many patients undergo this procedure in the main operating room under general anesthesia. These patients often have microscopic metastases in sentinel nodes that could be missed by histopathologic examination. Techniques of reverse transcriptase polymerase chain reaction (RT-PCR) could detect these metastases if the nodes could be preserved intraoperatively. STUDY DESIGN: Fifty patients with melanoma > or = mm thick underwent sentinel lymphadenectomy under local anesthesia in an outpatient surgical unit. Sentinel nodes were identified using blue dye and technetium-99 sulfur colloid and a hand-held gamma probe. Each node was sectioned, with half sent for routine histopathologic study and half preserved in liquid nitrogen. We used RT-PCR to detect mRNA for tyrosinase and Melanoma Antigen Recognized by T cells-1 (MART-1). RESULTS: All patients were able to tolerate sentinel lymph node biopsy under local anesthesia. Sentinel lymph nodes were obtained in 100% of our patients, and usable mRNA was harvested from all but five. Ten patients had positive sentinel node(s) by standard histopathologic examination, and all of these nodes were also positive for MART-1 and tyrosinase. Three patients with negative results by histopathology had positive results by RT-PCR analysis. The average cost of these outpatient operations was 38% less than the same operations performed in the main operating room under general anesthesia. CONCLUSIONS: Sentinel lymphadenectomy under local anesthesia in an outpatient setting and intraoperative lymph node preservation in liquid nitrogen are both feasible. Both tyrosinase and MART-1 are promising markers in the detection of occult melanoma in lymph nodes.

The amino terminus of Cdk2 binds p21.

The cyclin-dependent kinase (Cdk) inhibitor known as p21, which is transcriptionally regulated by p53, can induce G1 arrest when overexpressed and inhibit the kinase activity of a wide variety of cyclin-Cdk complexes. Previous studies have demonstrated that a portion of the conserved region of p21 (amino acids 46-78), which is homologous to similar regions in the related Cdk inhibitors p27 and p57, can bind to Cdk2, and that this region is essential for kinase inhibition. However, the site(s) on Cdk2 that are involved in p21 binding have not been identified. We therefore created mutant Cdk2 molecules with various N-terminal and C-terminal deletions and tested each for their ability to bind to p21 by the yeast two-hybrid and the double-tagging assays. None of the deletion mutants tested bound to p21 by either assay. We next tested whether p21 could bind to Cdk7, a component of the cyclin-activating kinase complex. By both the double-tagging and yeast two-hybrid assays, p21 failed to bind to this protein, consistent with previous reports. However, hybrid molecules consisting of the amino-terminal half of Cdk2 and the carboxy-terminal half of Cdk7 (Cdk2/Cdk7) could bind to p21 by both assays, whereas the Cdk7/Cdk2 hybrids could not. Furthermore, the yeast Cdc28 protein, which is 65% identical with Cdk2, failed to bind to p21 by both the yeast two-hybrid and double-tagging assays. Cdk2/Cdc28 hybrids but not Cdc28/Cdk2 hybrids could bind to p21. These results suggest that the amino-terminal half of Cdk2 is important for p21 binding, consistent with the recently published crystal-lographic data. Our data also suggest that the three-dimensional structure of Cdk2 is likely altered by creating deletion mutants from either the amino- or carboxy-terminal end of the protein. Finally, we have mutated the Cdc28/Cdk2 hybrid protein and isolated several mutants, which are able to bind to p21. This approach may be useful for identifying residues in Cdk2 and Cdc28 that affect their ability to bind to p21 and complement the crystallographic data.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

Screening for in vivo protein-protein interactions.

We describe an in vivo approach for the isolation of proteins interacting with a protein of interest. The protein of interest is "tagged" with a portion of the biotin carboxylase carrier protein (BCCP), encoded on a specially constructed plasmid, so that it becomes biotinylated in vivo. The "query" proteins (e.g., those in a cDNA library) are tagged by fusing them to the 3' end of the lacZ gene on a lambda vector in such a way that the beta-galactosidase activity is not disrupted. These phage are transfected into cells containing the plasmid encoding the BCCP-tagged protein. The infection lyses the cells and exposes the protein complexes. The BCCP-tagged protein and any associated protein(s) are "captured" by using avidin, streptavidin, or anti-biotin antibody-coated filters. The detection of bound protein is accomplished by directly assaying for beta-galactosidase activity on the filters. Positive plaques can be plaque-purified for DNA sequencing. We have tested this approach by using c-Fos and c-Jun as our model system. We show that avidin, streptavidin, or polyclonal anti-biotin (but not a monoclonal anti-biotin) antibody is capable of specifically capturing in vivo biotinylated beta-galactosidase and c-Jun and that this capture is dependent upon the presence of both avidin and the BCCP moiety. Further, complexes containing c-Jun and c-Fos can also be isolated in this manner, and the isolation of this complex is dependent on the presence of c-Fos, c-Jun, avidin, and the BCCP moiety. We discuss the possible uses and limitations of this technique for isolating proteins that interact with a known protein.

A contingent replication assay for the detection of protein-protein interactions in animal cells.

We have developed a sensitive and rapid assay system termed the contingent replication assay (CRA) for selecting cDNAs with desired functional properties from a cDNA library. The system functions in animal cells and permits enrichment of the desired cDNA in small-scale and convenient experiments. The assay can be used for the enrichment of proteins that activate transcription from conditional enhancers, bind to specific DNA sequences, or interact with target proteins of interest. In this communication we report the application of this assay to study protein-protein interactions in animal cells.

Characterization of cDNA for the large subunit of the transcription initiation factor TFIIF.

At least six chromatographically resolvable general transcription factors may participate in accurate initiation by RNA polymerase II in HeLa cell-derived systems. TFIIF (also termed FC, RAP30/74 and beta/gamma) can bind directly to RNA polymerase II in solution and decrease the affinity of RNA polymerase II for nonspecific DNA. From studies on the kinetics of transcription initiation, on the composition of transcription initiation complexes fractionated by acrylamide gel electrophoresis, and on template competition experiments, TFIIF is known to act at an intermediate stage in initiation complex formation. It acts after TFIID firmly associates with DNA, but coincidentally with or immediately after RNA polymerase II binding to DNA, and before the recruitment of factor TFIIE. TFIIF may or may not have DNA helicase activity. The small subunit (RAP30) of TFIIF has been cloned and shows some amino-acid sequence homology to bacterial sigma factors. We have partially sequenced the RAP74 protein from purified HeLa cells, cloned its complementary DNA and shown that its translation product can interact with RAP30 in vitro as well as in vivo. The cDNA predicts an amino-acid sequence that lacks obvious DNA or RNA helicase motifs. It has regions rich in charged amino acids, including segments containing a higher content of acidic amino acids than are found in strong transcriptional activators such as VP16.

Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase.

The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of approximately 200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.