Threonine phosphorylation of the MMAC1/PTEN PDZ binding domain both inhibits and stimulates PDZ binding.

Two-hybrid searches with the tumor suppressor MMAC1/PTEN isolated the proteins hDLG and hMAST205. Further two-hybrid analysis and microtiter plate binding assays localized the sites of interaction to PDZ domains from hDLG and hMAST205 and the PDZ binding domain at the COOH terminus of MMAC1/PTEN. A synthetic peptide derived from the MMAC1/PTEN PDZ binding domain (MMAC1/PTEN-PDZBD) was used to coprecipitate proteins from A431 human cell lysate. The recovered proteins were resolved by SDS-PAGE and immobilized on a nitrocellulose membrane. Treatment of this membrane with an anti-hDLG antibody identified a Mr 140,000 band, consistent with the size of hDLG. Treatment of this membrane with the MMAC1/PTEN-PDZBD peptide identified a single prominent band of slightly larger than Mr 200,000 (Mr 200,000 kDa). Threonine phosphorylation of the MMAC1/ PTEN-PDZBD peptide inhibited both microtiter plate binding to the hDLG and hMAST205 PDZ domains and coprecipitation of the Mr 140,000 and > 200,000 proteins, but promoted coprecipitation of proteins of approximately Mr 90,000 and Mr 120,000 from A431 cell lysate. This result suggests phosphorylation of the MMAC1/PTEN PDZ binding domain can both inhibit and promote PDZ interactions.

Polyphosphoinositides inhibit the interaction of vinculin with actin filaments.

Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin's head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P2) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P2 on the actin and talin-binding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P2.

Isolation of peptides from phage-displayed random peptide libraries that interact with the talin-binding domain of vinculin.

Peptides isolated from combinatorial libraries typically interact with, and thus help to characterize, biologically relevant binding domains of target proteins. To characterize the binding domains of the focal adhesion protein vinculin, vinculin-binding peptides were isolated from two phage-displayed random peptide libraries. Altogether, five non-similar vinculin-binding peptides were identified. Despite the lack of obvious sequence similarity between the peptides, binding and competition studies indicated that all five interact with the talin-binding domain of vinculin and do not disrupt the binding of alpha-actinin or paxillin to vinculin. The identified peptides and talin bind to vinculin in a comparable manner; both bind to immobilized vinculin, but neither binds to soluble vinculin unless the C-terminus of vinculin has been deleted. An analysis of amino acid variants of one of the peptides has revealed three non-contiguous motifs that also occur in the region of talin previously demonstrated to bind vinculin. Amino acid substitutions within a 127-residue segment of talin capable of binding vinculin confirmed the importance of two of the motifs and suggest that residues critical for binding are within a 16-residue region. This study demonstrates that the vinculin-binding peptides interact with vinculin in a biologically relevant manner and represent an excellent tool for further study of the biochemistry of vinculin.

Identification of calmodulin-binding peptide consensus sequences from a phage-displayed random peptide library.

The calcium-binding protein, calmodulin (CaM), was used to screen a phage library displaying random peptides 26 amino acids (aa) in length. Twenty CaM-binding peptides were identified, 17 of which contained one of three consensus sequence motifs: + W-OlambdaR, WRAAV or WRXXAAAL, where +, -, O, lambda and X are positively charged, negatively charged, hydrophobic, leucine or valine, and any residue, respectively. The Trp residue in these motifs is located within 14 aa of the N-terminus of the displayed peptide. Previous studies [Dedman et al., J. Biol. Chem. 268 (1993) 23025-23030] using a library displaying random peptides 15 aa in length identified CaM-binding peptides which contained a Trp-Pro dipeptide motif. These results suggest that the type of CaM-binding motif identified can vary between different types of combinatorial peptides.

Characterization of phage that bind plastic from phage-displayed random peptide libraries.

During routine screenings of random peptide libraries displayed at the N terminus of the pIII coat protein of M13 bacteriophage, clones were isolated that bound directly to the polystyrene (PS) surface used to immobilize the target protein. The plastic-binding phage (P-b phi) bind to both unblocked plastic (PS and polyvinyl chloride, PVC) and plastic blocked with bovine serum albumin (BSA) but require non-ionic detergent to bind to plastic blocked with milk. Comparison of the P-b phi to antibody-binding phage (Ab-b phi) indicates that similar numbers of phage particles are bound, but fewer P-b phi the recovered by acid elution. Sequence determination of the displayed peptides reveals they lack amino-acid sequence similarity yet are highly enriched for the Tyr and Trp residues. However, because not all phage that display peptides rich in Tyr and Trp residues bind to plastic, and other methods of screening random peptide libraries have identified different classes of plastic-binding peptides, the relative abundance of Tyr and Trp residues should not be considered diagnostic of plastic-binding. In summary, these results help characterize one of the most common methods used to screen random peptide libraries and suggest strategies to avoid isolating P-b phi. Furthermore, while it is generally believed that proteins bind to plastic by non-specific interactions, these results show that a bias in aa composition can exist.

Rodent L1 evolution has been driven by a single dominant lineage that has repeatedly acquired new transcriptional regulatory sequences.

All mammalian genomes contain approximately 100,000 copies of the transposable element LINES-1 (L1). Phylogenetic analysis indicates that the L1 progenitor predates the mammalian radiation; since that time, the open reading frames encoded in L1 have evolved under selection. The least conserved regions within L1 are the 5'-terminal transcriptional regulatory sequences. In rodents, four types of L1 elements (A, F, and V from mouse and R from rat) have been defined according to the type of apparently nonhomologous promoter sequence present at the 5' end. In this study, we investigate the relationships between these four types of promoters. DNA sequence was determined from approximately 1.5-kb regions from the 5' ends of seven F- and three V-type L1 elements. These sequences were aligned with 29 previously reported L1 elements. Phylogenetic analysis was then performed on the homologous regions of the alignment. The results indicate that in mouse all of the A-, F-, and V-type elements belong to a single dominant lineage but were inserted into the genome during different time periods; V-type elements are the oldest, while A-type elements are the most recently inserted. V-type elements also appear ancestral to the R-type elements found in rat and therefore were replicatively competent prior to the divergence of rat and mouse. Analysis of sequence identity indicates that the different 5' promoters did not derive from a common ancestor. Therefore, the dominant L1 lineage appears to have acquired novel promoter sequences from non-L1 sources. Transposable elements from a wide range of species show similar structural rearrangements, suggesting that acquisition of new sequences may be a common theme in their evolution.

Molecular resurrection of an extinct ancestral promoter for mouse L1.

The F-type subfamily of LINE-1 or L1 retroposons [for long interspersed (repetitive) element 1] was dispersed in the mouse genome several million years ago. This subfamily appears to be both transcriptionally and transpositionally inactive today and therefore may be considered evolutionarily extinct. We hypothesized that these F-type L1s are inactive because of the accumulation of mutations. To test this idea we used phylogenetic analysis to deduce the sequence of a transpositionally active ancestral F-type promoter, resurrected it by chemical synthesis, and showed that it has promoter activity. In contrast, F-type sequences isolated from the modern genome are inactive. This approach, in which the automated DNA synthesizer is used as a "time machine," should have broad application in testing models derived from evolutionary studies.

An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets.

We have examined the potential of isolating novel ligands from a library of M13 pIII-fusion phage displaying peptides composed of 38 random amino acids (aa). The library was panned with streptavidin (SA) and a polyclonal goat antimouse IgG Fc antibody (Ab) preparation coupled to paramagnetic beads. SA selected two classes of phage from the library. One class exhibited the aa motif, HP(Q/M) theta (where theta signifies a non-polar aa), similar to the motif identified by Devlin et al. [Science 249 (1990) 404-406] using a 15-aa random peptide library displayed on phage. The other class of phage had no discernible motif. In binding experiments, the non-HP(Q/M) theta phage had a slightly higher affinity for SA than did the motif phage. Both classes of SA-binding phage failed to bind native and non-glycosylated forms of avidin, even though SA and avidin are structurally similar and both proteins possess extraordinary affinities for biotin. The polyclonal goat anti-mouse IgG Fc Ab preparation selected phage displaying sequences similar to a region of the mouse IgG Fc. Thus, a single immunodominant epitope on the mouse IgG Fc was identified. Furthermore, a second phage displaying peptides with no discernible sequence similarities to mouse IgG Fc was isolated. Thus, an M13 library displaying 38-aa peptides can yield phage with affinity for various targets. Finally, we have observed a biological bias against odd numbers of Cys residues in the displayed peptides.

L1 A-monomer tandem arrays have expanded during the course of mouse L1 evolution.

LINE-1 (L1) is a family of highly repeated DNA sequences interspersed throughout the mammalian genome. Individual L1 elements are thought to be generated by a transposition mechanism involving reverse transcription of an RNA intermediate followed by insertion into a new genomic site. In mice, three major families of L1 elements, termed "A," "F," and "V," have been defined on the basis of the sequence found at the 5' terminus. Previous analyses of A-monomers have demonstrated sequence heterogeneity among individual A-monomers, variation in the length of A-monomer sequences, and the presence of transcriptional regulatory activity. To provide a detailed characterization of A-monomers as a foundation for studying their transcriptional regulatory activity, we have analyzed the sequences of 39 complete or partial length A-monomers from 20 different mouse L1 elements. A-monomers can be classified into six different types according to shared-sequence length variations. Consensus sequences for the six types of A-monomers indicate conservation of possible transcription factor-binding sequences. Specific subgroups of A-monomers correlate with the relative dispersal time of a mouse L1 element. A phylogenetic analysis of A-monomers indicates that the length variants represent good diagnostic sites for phylogenetic subgroups of A-monomer sequences. These observations suggest a model for the evolution of A-monomer tandem arrays that involves stepwise mutation and array expansion in the 5' direction. Hybridization data provide a minimum estimate of 16,000 copies of the A-monomer sequence in the mouse haploid genome, with an average array length of 2.1 monomer units.

Composite of A and F-type 5' terminal sequences defines a subfamily of mouse LINE-1 elements.

The 5' terminus of full-length L1 elements contains transcriptional control sequences. In mouse L1 (L1Md) elements, these sequences exist as an array of tandem direct repeats. Two types of repeat units, termed A-monomers and F-monomers, have been reported. Both monomers are about 200 bp in length but share no significant sequence homology. Previous studies have identified L1Md elements containing either A or F-monomers but not both. Here we describe three "composite" L1Md elements that contain both types of monomer sequence. Two of these composite L1Md elements are highly homologous and share the same structural rearrangements, implying that they arose from a common ancestor that has the same composite 5' end.

Nucleotide sequence of the BALB/c mouse beta-globin complex.

The nucleotide sequence of 55,856 base-pairs containing all seven beta-globin homologous structures from chromosome 7 of the BALB/c mouse is reported. This sequence links together previously published sequences of the beta-globin genes, pseudogenes and repetitive elements. Using low stringency computer searches, we found no additional beta-globin homologous sequences, but did find many more long interspersed repetitive sequences (L1) than predicted by hybridization. L1 is a major component of the mouse beta-globin complex with at least 15 elements comprising about 22% of the reported sequence. Most open reading frames greater than 300 base-pairs in the cluster overlap with L1 repeats or globin genes. Polypurine, polypyrimidine and alternating purine/pyrimidine tracts are not evenly dispersed throughout the complex, but they do not appear to be excluded from or restricted to particular regions. Several regions of intergenic homology were detected in dot-plot comparisons of the mouse sequence with itself and with the human beta-globin sequence. The significance of these homologies is unclear, but these regions are candidates for further study in functional assays in erythroid cell lines or transgenic animals.