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Unregulated expression of either the retinoblastoma protein (pRB) or the related protein p107 can cause growth arrest of sensitive cells in the G1 phase of the cell cycle. However, growth arrests mediated by p107 and pRB are not identical. Through structure‐function and co‐expression analyses we have dissected the p107 molecule into two domains that independently are able to block cell cycle progression. One domain corresponds to the sequences needed for interaction with the transcription factor E2F, and the other corresponds to the interaction domain for cyclin A or cyclin E complexes. In cervical carcinoma cell line C33A, which was previously shown to be sensitive to p107 but resistant to pRB growth suppression, only the cyclin binding domain is active as a growth suppressor. Furthermore, we show that these two independent domains are functional in untransformed mouse fibroblasts. Together, these results provide experimental evidence for the presence of two functional domains in p107 and pinpoint an important functional difference between p107 and pRB.

The cellular tumour antigen p53 is implicated in the transformation process. To compare p53 from transformed cells and their normal counterparts in detail, and so to identify any structural differences that might alter p53 function, requires information on the primary structure of the protein. By making use of immunochemical techniques we have been able to purify nanomole amounts of p53. This was sufficient, using high sensitivity automated gas‐phase sequencing techniques to determine the amino acid sequence of two tryptic peptides from p53. Their sequences agree completely with the predicted polypeptide sequence derived from a cloned cDNA for p53 mRNA and provide the first data on the amino acid sequence of p53. A combination of the high sensitivity amino acid sequencing procedures used here and cDNA sequence analysis should provide the complete amino acid sequence of p53.

The protein product of the retinoblastoma (RB) gene is thought to function in a pathway that restricts cell proliferation. Recently, transforming proteins from three different classes of DNA tumor viruses have been shown to form complexes with the RB protein. Genetic studies suggest that these interactions with the RB protein are important steps in transformation by these viruses. In order to understand better the function of the RB‐viral oncoprotein complexes, we have mapped the regions of the RB protein that are necessary for these associations. Two non‐contiguous regions of RB were found to be essential for complex formation with adenovirus E1A or SV40 large T antigen. These two regions are found between amino acids 393 and 572 and 646 and 772. Interestingly, these binding sites on RB overlap with the positions of naturally occurring, inactivating mutations of the RB gene. These results strongly suggest that these viral oncoproteins are targeting a protein domain that is an important site in the normal function of the RB protein.

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.

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.

In addition to the protein products of the adenovirus E1A 13S and 12S mRNAs, monoclonal antibodies specific for the E1A proteins immunoprecipitate polypeptides with relative mol. wt of 30,000 (30 kd) and 35,000 (35 kd) from extracts of infected cells. The 30 kd and 35 kd proteins are encoded by novel mRNAs referred to as the 10S and 11S mRNAs, respectively. These two mRNAs arise from differential splicing of the E1A precursor RNA. For the 10S mRNA, the precursor is spliced twice, once removing the region between nucleotides 637 and 854 and once between 974 and 1229. The splice between nucleotides 974 and 1229 is identical to the one used for the processing of the 12S mRNA. Synthesis of the 11S mRNA also utilizes two splicing events. One of these is identical to the 637/854 splice of the 10S mRNA, and the other removes the region between nucleotides 1112 and 1229, a splice junction also found in the 13S mRNA. All four mRNAs used the same reading frame and, therefore, code for related proteins. The products of the 10S and 11S mRNAs are identical to the products of the 12S and 13S mRNAs, respectively, except for an internal stretch of 27 amino acids removed by the 637/854 splice. Within this segment is a group of amino acid residues that is highly conserved between different adenovirus serotypes. Mutant adenoviruses in which the wild‐type E1A sequences have been replaced with cDNA copies of the 10S or 11S mRNAs are defective for growth on HeLa cells suggesting that this region is important for viral growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of the retinoblastoma protein (pRB) is assumed to regulate its growth‐controlling function. Moreover, hypophosphorylated and hyperphosphorylated forms of pRB can be distinguished by virtue of the distinct affinities with which they bind to the cell nucleus. This property allows the identification of individual cell nuclei that contain pRB in one or the other form. We show here that after cells emerge from a quiescent (G0) state, conversion of their complement of pRB into a hyperphosphorylated form occurs in late G1, preceding entry into S phase by several hours. Thus, contrary to earlier reports, pRB phosphorylation is not co‐ordinated with the G1‐S transition and may not directly regulate it. A distinct set of phosphopeptides is found exclusively in those forms of pRB that show the loose nuclear association characteristic of the hyperphosphorylated form of pRB. Another set of phosphopeptides is found with both hypophosphorylated and hyperphosphorylated forms. This suggests the existence of distinct patterns of phosphorylation that are associated with different subsets of pRB molecules. We conclude that substantial phosphorylation of pRB exists in G1 even prior to the hyperphosphorylation point. Cyclin‐dependent kinases can cause a liberation of pRB from cell nuclei in vitro. Phosphorylation by members of this kinase family is therefore likely to be directly involved in the change in nuclear affinity in vivo and the associated changes in pRB functioning.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2‐related protein is a major pRB kinase.