Ed Harlow

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105‐RB). Similar to the E7 protein of HPV‐16, the E7 proteins of HPV‐18, HBV‐6b and HPV‐11 were found to associate with p105‐RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV‐16 and HPV‐18) formed complexes with p105‐RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105‐RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV‐1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105‐RB. The amino acid sequences of the HPV‐16 E7 protein involved in complex formation with p105‐RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105‐RB. Furthermore, the HPV‐16 E7‐p105‐RB complex was detected in an HPV‐16‐transformed human keratinocyte cell line.

The retinoblastoma protein is phosphorylated during specific phases of the cell cycle

p105-RB is the product of the retinoblastoma tumor suppressor gene. It is a nuclear phosphoprotein hypothesized to act as an inhibitor of cellular proliferation, yet surprisingly it is present in actively dividing cells. To look for changes in p105-RB that may regulate its activity during the cell cycle, we generated synchronized cell populations and followed their progression through the cell cycle. p105-RB is synthesized throughout the cycle, but is phosphorylated in a phase-specific manner. In the G0 and G1 phases of the cell cycle, an unphosphorylated species of the protein is the only detectable form, whereas in the S and G2/M phases, multiple phosphorylated species of p105-RB are detected.

A family of human cdc2-related protein kinases.

The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2‐related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins.

Tumor Induction and Tissue Atrophy in Mice Lacking E2F-1

The retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor that regulates gene expression by physically associating with transcription factors such as E2F family members. Although pRB and its upstream regulators are commonly mutated in human cancer, the physiological role of the pRB-E2F pathway is unknown. To address the function of E2F-1 and pRB/E2F-1 complexes in vivo, we have produced mice homozygous for a nonfunctional E2F-1 allele. Mice lacking E2F-1 are viable and fertile, yet experience testicular atrophy and exocrine gland dysplasia. Surprisingly, mice lacking E2F-1 develop a broad and unusual spectrum of tumors. Although overexpression of E2F-1 in tissue culture cells can stimulate cell proliferation and be oncogenic, loss of E2F-1 in mice results in tumorigenesis, demonstrating that E2F-1 also functions as a tumor suppressor.

Identification of G1 kinase activity for cdk6, a novel cyclin D partner.

A family of vertebrate cdc2-related kinases has been identified, and these kinases are candidates for roles in cell cycle regulation. Here, we show that the human PLSTIRE gene product is a novel cyclin-dependent kinase, cdk6. The cdk6 kinase is associated with cyclins D1, D2, and D3 in lysates of human cells and is activated by coexpression with D-type cyclins in Sf9 insect cells. Furthermore, we demonstrate that endogenous cdk6 from human cell extracts is an active kinase which can phosphorylate pRB, the product of the retinoblastoma tumor suppressor gene. The activation of cdk6 kinase occurs during mid-G1 in phytohemagglutinin-stimulated T cells, well prior to the activation of cdk2 kinase. This timing suggests that cdk6, and by analogy its homolog cdk4, links growth factor stimulation with the onset of cell cycle progression.

The retinoblastoma tumour suppressor in development and cancer

Since its discovery, the retinoblastoma (RB) tumour-suppressor protein has been a focal point of cancer research. Accumulating evidence indicates a complex role for RB in cell proliferation, differentiation and survival. To further complicate matters, proteins that are related to RB have redundant as well as antagonistic functions. Recent studies of knockout mice and cells that lack one or more of these proteins have begun to clarify their various context-specific functions and the unique activity of this tumour suppressor.

A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F

The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3, was identified by the ability of its gene product to interact with pRB. RBP3 bound to pRB both in vitro and in vivo, and this binding was competed by viral proteins known to disrupt pRB-E2F association. RBP3 bound to E2F recognition sequences in a sequence-specific manner. Furthermore, transient expression of RBP3 caused a 10-fold transactivation of the adenovirus E2 promoter, and this transactivation was dependent on the E2F recognition sequences. These properties suggest that RBP3 encodes E2F, or an E2F-like protein.

Cellular targets for transformation by the adenovirus E1A proteins

Three cellular proteins, including species of 300,000 daltons and 107,000 daltons as well as p105-RB, the product of the retinoblastoma susceptibility gene, stably interact with the adenovirus E1A proteins. To help determine the functional basis of these interactions, the regions of E1A that participate in these interactions were mapped using a series of deletion mutants. The 300,000 dalton and the 107,000 dalton proteins interacted with sequences within amino acids 1 to 76 and 121 to 127, respectively. Interaction with the third cellular protein, p105-RB, required the presence of sequences from two noncontiguous regions of the E1A polypeptide chain, amino acids 30 to 60 and 121 to 127. The regions of E1A that are required for these interactions coincided precisely with the regions of E1A that are required for its transforming function. These results suggest that the interactions with these cellular proteins are fundamental to the transforming activity of E1A.

Inhibition of E2F-1 transactivation by direct binding of the retinoblastoma protein.

Loss of a functional retinoblastoma tumor suppressor gene product, pRB, is a key step in the development of many human tumors. pRB is a negative regulator of cell proliferation and appears to participate in control of entry into the S phase of the cell cycle. The recent demonstration that pRB binds to transcription factor E2F has provided a model for the mechanism of pRB-mediated growth regulation. Since adenovirus E1A proteins dissociate the pRB-E2F complexes and stimulate E2F-dependent transcription, it has been suggested that pRB inhibits E2F transactivation. Although some evidence for this hypothesis has been provided, it has not been possible to determine the mechanism of pRB-mediated inhibition of E2F transactivation. In this study, we constructed mutants of E2F-1 that do not bind to pRB yet retain the ability to transactivate the adenovirus E2 promoter through E2F DNA-binding sites. We demonstrated that transactivation mediated by the wild-type E2F-1 protein was inhibited by overexpression of wild-type pRB but not by a naturally occurring mutant of pRB. Transactivation mediated by mutants of E2F-1 which do not bind to pRB was not affected by overexpression of wild-type pRB. Furthermore, when the E2F-1 transactivation domain was fused to the GAL4 DNA-binding domain, pRB inhibited GAL4-E2F-1 transactivation through GAL4 sites. Expression of pRB did not inhibit transactivation mediated by GAL4-E2F-1 mutant constructs which were devoid of pRB binding. In conclusion, these data demonstrate that pRB inhibits E2F-dependent transactivation by direct protein-protein interaction.

The retinoblastoma protein binds to a family of E2F transcription factors.

E2F is a transcription factor that helps regulate the expression of a number of genes that are important in cell proliferation. Recently, several laboratories have isolated a cDNA clone that encodes an E2F-like protein, known as E2F-1. Subsequent characterization of this protein showed that it had the properties of E2F, but it was difficult to account for all of the suggested E2F activities through the function of this one protein. Using low-stringency hybridization, we have isolated cDNA clones that encode two additional E2F-like proteins, called E2F-2 and E2F-3. The chromosomal locations of the genes for E2F-2 and E2F-3 were mapped to 1p36 and 6q22, respectfully, confirming their independence from E2F-1. However, the E2F-2 and E2F-3 proteins are closely related to E2F-1. Both E2F-2 and E2F-3 bound to wild-type but not mutant E2F recognition sites, and they bound specifically to the retinoblastoma protein in vivo. Finally, E2F-2 and E2F-3 were able to activate transcription of E2F-responsive genes in a manner that was dependent upon the presence of at least one functional E2F binding site. These observations suggest that the E2F activities described previously result from the combined action of a family of proteins.