Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway.

PTEN/MMAC1 is a tumor suppressor gene located on chromosome 10q23. Inherited PTEN/MMAC1 mutations are associated with a cancer predisposition syndrome known as Cowden's disease. Somatic mutation of PTEN has been found in a number of malignancies, including glioblastoma, melanoma, and carcinoma of the prostate and endometrium. The protein product (PTEN) encodes a dual-specificity protein phosphatase and in addition can dephosphorylate certain lipid substrates. Herein, we show that PTEN protein induces a G1 block when reconstituted in PTEN-null cells. A PTEN mutant associated with Cowden's disease (PTEN;G129E) has protein phosphatase activity yet is defective in dephosphorylating inositol 1,3,4,5-tetrakisphosphate in vitro and fails to arrest cells in G1. These data suggest a link between induction of a cell-cycle block by PTEN and its ability to dephosphorylate, in vivo, phosphatidylinositol 3,4,5-trisphosphate. In keeping with this notion, PTEN can inhibit the phosphatidylinositol 3,4, 5-trisphosphate-dependent Akt kinase, a downstream target of phosphatidylinositol 3-kinase, and constitutively active, but not wild-type, Akt overrides a PTEN G1 arrest. Finally, tumor cells lacking PTEN contain high levels of activated Akt, suggesting that PTEN is necessary for the appropriate regulation of the phosphatidylinositol 3-kinase/Akt pathway.

Cell density modulates protein-tyrosine phosphorylation.

The growth of normal cells is arrested at saturating cell density in a process termed contact inhibition. An understanding of how cells communicate their contact with one another is critical for determining how cancers develop and spread. Because the molecular details of how fibroblasts communicate density changes are unclear, we examined cell density itself as a source of signaling events rather than examine specific receptors. A technique was developed to measure tyrosine phosphorylation acutely as a function of cell density. The tyrosine phosphorylation of a number of proteins was found to be modified in response to cell density. Three of these proteins were identified as Src, paxillin, and focal adhesion kinase (FAK), all of which show an increase in their tyrosine phosphate levels with increasing density. All of these proteins are found in focal adhesions, and both FAK and paxillin are believed to be localized exclusively in focal adhesions. Thus, changing cell density alters tyrosine phosphorylation of focal adhesion components.

Characterization of the polyomavirus late polyadenylation signal.

The polyomavirus late polyadenylation signal is used inefficiently during the late phase of a productive viral infection. Inefficient polyadenylation serves an important purpose for viral propagation, as it allows a splicing event that stabilizes late transcripts (G. R. Adami, C. W. Marlor, N. L. Barrett, and G. G. Carmichale, J. Virol. 63:85-93, 1989; R. P. Hyde-DeRuyscher and G. G. Carmichael, J. Virol. 64:5823-5832, 1990). We have recently shown that late-strand readthrough transcripts serve as natural antisense molecules to downregulate early-strand RNA levels at late times in infection (Z. Liu, D. B. Batt, and G. G. Carmichael, Proc. Natl. Acad. Sci. USA 91:4258-4262, 1994). Thus, poor polyadenylation contributes to the early-late switch by allowing the formation of more stable late RNAs and by forming antisense RNA to early RNAs. The importance of late poly(A) site inefficiency in the viral life cycle has prompted us to map the cis elements of this site. Since the polyomavirus late site proved a poor substrate for in vitro polyadenylation, we used an in vivo assay which allowed us to map the cis sequences required for its function. In this assay, various fragments containing the AAUAAA and different surrounding sequences were placed 1.4 kb upstream of a second, wild-type signal. The second signal served to stabilize transcripts that are not processed at the upstream site, allowing accurate quantitation of relative poly(A) site use by an RNase protection assay. Processing was primary at the upstream site when a large fragment surrounding the poly(A) signal (50 nucleotides [nt] upstream and 90 nt downstream) was tested in this assay, demonstrating that this fragment contains the essential cis elements. Deletion analysis of this fragment revealed that most but not all upstream sequences can be removed with little effect on polyadenylation efficiency, indicating the absence of a strong stimulatory upstream element. Deletion of all but 25 nt downstream of the AAUAAA reduced polyadenylation activity only by half, demonstrating that processing can occur at this site despite the lack of downstream sequences. Thus, the core cis element for polyadenylation is quite small, with most important cis-acting elements lying within 19 nt upstream and 25 nt downstream of the AAUAAA sequence. This core contains the AAUAAA hexanucleotide, an upstream A/U-rich element, and three identical repeats of a 6-nt sequence, UAUUCA. Polyadenylation was eliminated or greatly reduced when either the AAUAAA or the three repeats were mutated.

Polyadenylation and transcription termination in gene constructs containing multiple tandem polyadenylation signals.

The processes of pre-mRNA 3'-end cleavage and polyadenylation have been closely linked to transcription termination by RNA polymerase II. We have studied the relationship between polyadenylation and transcription termination in gene constructs containing tandem poly(A) signals, at least one of which is the inefficient polyomavirus late poly(A) site. When identical tandem viral signals were separated by fewer than 400 bp, they competed for polyadenylation. The upstream site was always chosen preferentially, but relative site choice was influenced by the distance between the signals. All of these constructs showed the same low level of transcription termination as wild type polyomavirus, which contains a single late poly(A) site. When tandem poly(A) signals were not identical, a stronger downstream signal could outcompete a weaker upstream signal for polyadenylation without altering the efficiency of transcription termination characteristic for use of the upstream signal. Thus, if a weak polyoma virus late poly(A) signal (associated with inefficient transcription termination) preceded a strong rabbit beta-globin signal (associated with efficient transcription termination), termination remained inefficient, but the distal signal was most often chosen for polyadenylation. These results are consistent with independent regulation of polyadenylation and transcription termination in this system and are discussed in light of current models for the dependence of transcription termination on a functional poly(A) site.

Targeted nuclear antisense RNA mimics natural antisense-induced degradation of polyoma virus early RNA.

We describe a general antisense strategy to inhibit target gene expression. The substitution of a cis-acting ribozyme for a polyadenylylation signal in an antisense expression vector results in the nuclear retention of RNAs and the efficient degradation of their targets. We demonstrate the utility of this system in polyoma virus, where early-strand RNA levels are downregulated in the nucleus by antisense late-strand counterparts. We show that mutations destabilizing these naturally occurring antisense transcripts lead to increased levels of early-strand RNAs. Furthermore, expression in trans of nuclear antisense transcripts lowers early-strand RNA levels and quantitatively mimics the natural regulation.

Splice site selection in polyomavirus late pre-mRNA processing.

Polyomavirus late pre-mRNAs contain one 5' splice site and two message body 3' splice sites, which are not used at equal frequencies. As a result of alternative splicing, the total late mRNA population consists of about 5% mVP2 (no message body splice chosen), about 15% mVP3 (promoter-proximal 3' splice site chosen), and about 80% mVP1 (promoter-distal 3' splice site chosen). To determine whether it is splice site strength that determines the ratio of spliced products, constructs containing duplicated or rearranged 3' splice sites were created. In construct VP1,1, 160 bp surrounding the VP3 3' splice site was substituted with the corresponding region of the VP1 3' splice site. This construct resulted in the duplication of the VP1 3' splicing signal. VP3,3 (two identical VP3 3' splice sites) and VP1,3 (VP1 and VP3 3' splice sites reversed) were similarly created. Each construct maintained wild-type spacing between the 3' splice sites. Analysis of RNAs from transfections showed that in each construct, the 3' splice closest to the polyadenylation site was used preferentially. Analysis of a number of additional constructs indicated that there are no strong cis-acting positive or negative regulators of polyomavirus late splicing; rather, splicing choices appear to be determined largely by relative position of splice sites.