cdk6 can shorten G(1) phase dependent upon the N-terminal INK4 interaction domain.

Deregulated activity of cdk4 or cdk6 can lead to inappropriate cellular proliferation and tumorigenesis accompanied by unchecked inactivation of the retinoblastoma tumor suppressor protein. Certain tumor types preferentially activate either cdk4 or cdk6, suggesting that these kinases may not be equivalently oncogenic in all cell types. Although it is clear that cdk4 can act as an oncogene at least in part by evading inhibition by p16(INK4a), the role of cdk6 in tumorigenesis is less well understood. To investigate the consequences of aberrant expression of cdk6, the requirements for proliferation caused by cdk6 overexpression were studied. cdk6-transfected U2OS cells displayed an accelerated progression through G(1) phase that was dependent on kinase activity and that did not correlate with p27 binding. Furthermore, a mutation that prevents cdk6 interaction with INK4 proteins (cdk6R31C) was found to inactivate the proliferative effect of cdk6 and increase cytoplasmic localization, despite the fact that this mutant could phosphorylate the retinoblastoma protein in vitro. Together, these data suggest a role for the cdk6 INK4 interaction domain in the generation of functional, nuclear cdk6 complexes and demonstrate the importance of elevated cdk6 kinase activity in G(1) acceleration.

Functional interaction of a novel cellular protein with the papillomavirus E2 transactivation domain.

The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.

Sequences flanking the core DNA-binding domain of bovine papillomavirus type 1 E2 contribute to DNA-binding function.

We have compared a series of molecular constructs that contain the minimal DNA-binding and dimerization domain of bovine papillomavirus type 1 (BPV-1) E2 alone or this binding domain plus the adjacent 16 or 40 amino acids to test the role of the flanking sequences in E2 function. The presence of these sequences resulted in an up to eightfold increase in the affinity of E2 for its target DNA and stabilized the protein against denaturation both in the absence of DNA and in the form of DNA-protein complexes. In addition, an aspartic acid-to-tyrosine mutation within the flanking region blocked DNA binding and function. These data demonstrate that sequences flanking the core domain contribute to E2 function and are, in fact, an integral part of the DNA-binding domain of BPV-1 E2.

Transcriptional activation function is not required for stimulation of DNA replication by bovine papillomavirus type 1 E2.

Bovine papillomavirus type 1 replication was previously shown to require both the E1 initiator protein and the E2 transactivator protein. We show here that E1, in the absence of E2, is sufficient for low-level bovine papillomavirus type 1 DNA replication in C-33A cells. In addition, studies of genetically isolated E2 point mutants demonstrate that enhancement of replication by E2 does not require its transcriptional activation function. The uncoupling of the E2 functions suggests that stimulation of transcription and replication by enhancer proteins occurs via divergent mechanisms.

Genetic analysis of the bovine papillomavirus E2 transcriptional activation domain.

The bovine papillomavirus type 1 E2 transactivator has a large amino-terminal 215-residue transcriptional activation domain (TAD) that is active in Saccharomyces cerevisiae and higher eukaryotic cells. Comparison to other transcriptional activators suggests that its functions may be mediated in part through two acidic regions, A1 and A2, in this domain. We have characterized the functional elements within the E2 TAD using LexA-E2 fusions and by screening randomly generated libraries of E2 mutations for transcriptional activation in yeast. The A1 region was highly sensitive to substitutions that reduce negative charge, although there was not a perfect correlation between overall charge and transcriptional activity. Mutations were isolated within a hydrophobic amino acid motif that overlaps the A2 region and resembles elements described in other viral and cellular transactivation domains. When fused to the LexA DNA binding domain, this hydrophobic motif within the acidic A2 region was unable to activate transcription in S. cerevisiae. Multiple highly defective mutations primarily altering hydrophobic amino acids were identified in the distal third of the E2 TAD. The transcription phenotype of many of these E2 TAD mutations was similar in yeast and COS cells.

The BPV-1 E2 DNA-contact helix cysteine is required for transcriptional activation but not replication in mammalian cells.

The papillomavirus E2 protein contains an amino-terminal region thought necessary and sufficient to support transcriptional activation and a carboxy-terminal region shown to direct sequence-specific DNA binding and dimerization. A cysteine residue in the center of the E2 DNA recognition helix is highly conserved among papillomavirus E2 proteins. Mutations of this cysteine in bovine papillomavirus type 1 E2 to serine and glycine resulted in proteins which failed to activate E2-dependent promoters in mammalian cells. These E2 mutants were DNA-binding competent, dimeric, and nuclear. When fused to the VP16 transactivation domain, C-terminal regions of E2 containing the mutations at 340 supported transcriptional activation, indicating that the heterologous trans-activation domain did not require cysteine in the DNA-binding helix as did the full-length E2 transactivating protein. Although cysteine-340 was required for transcriptional activation it was not required for DNA replication in vivo. Together, these results suggest that the E2 DNA-binding domain may directly contribute to functions of transcriptional activation previously thought limited to the N-terminal domain.

Drosophila Krüppel protein is a transcriptional repressor.

Krüppel (Kr), one of the zygotically active Drosophila segmentation genes, is expressed in a restricted domain during the blastoderm stage of embryogenesis and is involved in the control of development of the thoracic and abdominal segments of the fly. Kr encodes a polypeptide containing DNA-binding zinc-finger motifs, disruptions of which yield Kr mutants. We have assayed the transcriptional activities of wild-type Kr protein as well as Lac repressor/Kr fusion proteins in HeLa and CV-1 cells. Wild-type Kr and a Lac-Kr chimaeric protein repressed transcription from reporter promoters in which a consensus Kr binding site derived from sequences within the even-skipped promoter had been inserted in an upstream position. We mapped the repression function of Kr to an alanine-rich amino-terminal region of the protein, as a Lac/Kr fusion protein containing only amino acids 26-110 of Kr repressed transcription from a reporter promoter containing upstream lac operators. This demonstrates that the DNA-binding and repression activities of the Kr protein are distinct. These data are consistent with genetic evidence that Kr represses even-skipped and hunchback expression, and suggest that Kr is a negative regulator of transcription in Drosophila.