D C Heimbrook

Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F.

E2F is a mammalian transcription factor that appears to play an important role in cell cycle regulation. While at least two proteins (E2F-1 and DP-1) with E2F-like activity have been cloned, studies from several laboratories suggest that additional homologs may exist. A novel protein with E2F-like properties, designated E2F-2, was cloned by screening a HeLa cDNA library with a DNA probe derived from the DNA binding domain of E2F-1 (K. Helin, J. A. Lees, M. Vidal, N. Dyson, E. Harlow, and A. Fattaey, Cell 70:337-350, 1992). E2F-2 exhibits overall 46% amino acid identity to E2F-1. Both the sequence and the function of the DNA and retinoblastoma gene product binding domains of E2F-1 are conserved in E2F-2. The DNA binding activity of E2F-2 is dramatically enhanced by complementation with particular sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified components of HeLa cell E2F, and anti-E2F-2 antibodies cross-react with components of purified HeLa cell E2F. These observations are consistent with a model in which E2F binds DNA as a heterodimer of two distinct proteins, and E2F-2 is functionally and immunologically related to one of these proteins.

Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein.

Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRBs ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.

An SH3 domain is required for the mitogenic activity of microinjected phospholipase C-γ1

Phospholipase activity is elevated in dividing cells. In response to growth factor stimulation, phospholipase C‐γ (PLC‐γ) binds to activated tyrosine kinase receptors via SH2 binding domains, resulting in phosphorylation of PLC‐γ and activation of its enzyme activity. These observations suggest that PLC‐γ participates in the signal transduction pathway employed by growth factors to promote mitogenesis. Consistent with this hypothesis, microinjection of purified bovine PLC‐γ into quiescent fibroblasts has been previously reported to initiate a mitogenic response [Smith et al. (1989) Proc. Natl. Acad. Sci. 86, 3659]. We have reproduced this result using recombinant rat PLC‐γ protein. Surprisingly, however, a catalytically inactive mutant of PLC‐γ, H335Q, also elicited a full mitogenic response. The capacity to induce mitogenesis by microinjection of PLC‐γ was mapped to the ‘Z’ domain of the protein, which contains PLC‐γs SH2 and SH3 motifs. Inactivation of the phosphorylated tyrosine binding properties of both SH2 domains had no effect on the mitogenic activity of the Z‐domain peptide. However, deletion of the SH3 domain resulted in a complete loss of activity. These results suggest that PLC‐γs mitogenic properties do not require the enzymes phospholipase activity, but are instead mediated by a novel pathway for mitogenic stimulation which is dependent upon an intact SH3 domain.

Therapeutic intervention and signaling

Significant advances in our understanding of intracellular signal transduction pathways have emerged within the past several years. It is now apparent that, under certain circumstances, particular isoforms of Ras can be prenylated by geranylgeranyl protein transferase as well as farnesyl protein transferase. New pathways controlling growth factor-dependent inhibition of apoptosis involving phosphoinositide 3-hydroxykinase and the protein kinase Akt have also been clarified.

Anions Modulate the Potency of Geranylgeranyl-Protein Transferase I Inhibitors

We have identified and characterized potent and specific inhibitors of geranylgeranyl-protein transferase type I (GGPTase I), as well as dual inhibitors of GGPTase I and farnesyl-protein transferase. Many of these inhibitors require the presence of phosphate anions for maximum activity against GGPTase Iin vitro. Inhibitors with a strong anion dependence were competitive with geranylgeranyl pyrophosphate (GGPP), rather than with the peptide substrate, which had served as the original template for inhibitor design. One of the most effective anions was ATP, which at low millimolar concentrations increased the potency of GGPTase I inhibitors up to several hundred-fold. In the case of clinical candidate l-778,123, this increase in potency was shown to result from two major interactions: competitive binding of inhibitor and GGPP, and competitive binding of ATP and GGPP. At 5 mm, ATP caused an increase in the apparent K d for the GGPP-GGPTase I interaction from 20 pm to 4 nm, resulting in correspondingly tighter inhibitor binding. A subset of very potent GGPP-competitive inhibitors displayed slow tight binding to GGPTase I with apparent on and off rates on the order of 106 m − 1s− 1 and 10− 3s− 1, respectively. Slow binding and the anion requirement suggest that these inhibitors may act as transition state analogs. After accounting for anion requirement, slow binding, and mechanism of competition, the structure-activity relationship determined in vitro correlated well with the inhibition of processing of GGPTase I substrate Rap1a in vivo.

ONCOGENE PRODUCTS AS THERAPEUTIC TARGETS FOR CANCER

Enormous progress has been made in the last several years in delineating signal transduction pathways associated with cell proliferation and apoptosis. The components of these pathways, which include both oncogenes and tumor suppressors, may provide viable targets for therapeutic intervention for the treatment of cancer and other diseases. This review highlights some of these recent biologic and pharmacologic advances, focusing on the ras pathway and on p53-dependent apoptosis.

Protein kinase inhibitors for the treatment of cancer

Protein kinases are integrated into nearly every facet of the regulation of mammalian cellular proliferation. The identification of particular kinase isoforms and tissuespecific expression affords the possibility of precise targeting of specific regulatory pathways if potent, highly selective, cell-penetrable inhibitors can be developed. At the very least, such inhibitors would provide opportunities to dissect complex interconnected regulatory pathways in intact cells, and sustain the promise of the development of therapeutic leads. This paper reviews recent developments in the identification of protein kinase targets for the treatment of cancer, and in the design of selective inhibitors for these enzymes.

Cloning and mutagenesis of the p110α subunit of human phosphoinositide 3′-hydroxykinase

Abstract Activation of phosphoinositide 3′-hydroxykinase (PI3K) is required for mitogenic signal transduction by several growth factors and oncogenes. PI3K is a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit. In the current study, we report the cloning and characterization of the p110α catalytic subunit of human PI3K. This clone is highly homologous (>99% amino acid identity) to bovine brain p110α, but contains 10 amino acid differences from the human p110α sequence previously reported. Comparison of this sequence with known Ser/Thr kinases and p110 homologs highlighted several conserved residues within the putative kinase domain. Mutational analysis of these residues (Asp 915 , (Asp 933 + Phe 934 ) yielded PI3K mutants with virtually complete loss of phosphoinositide phosphorylating activity. Expression of the wild-type p110α protein in CHO cells is sufficient to activate the serum response element derived from the promoter of c- fos , an immediate early gene product. In contrast, the catalytically impaired p110α mutants as well as the p85α subunit of PI3K were inactive in the fos assay. These studies suggest that the mitogenic signal transduction pathway mediated by PI3K is dependent upon the enzymatic activity of the p110α subunit of PI3K.