Koichi Okumura

PCAF Modulates PTEN Activity

The PTEN protein has a single catalytic domain possessing both lipid phosphoinositol and protein phosphatase activities. The lipid phosphoinositol phosphatase activity is essential for PTEN to block the cell cycle in the G1 phase and thereby to suppress tumor formation and progression (Cantley, L. C., and Neel, B. G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4240-4245), although the mechanisms governing PTEN activity under normal and neoplastic growth conditions remain unclear. Here, we report that PTEN interacts physically and functionally with PCAF, a histone acetyltransferase that regulates gene transcription through interaction with p300/CBP and various sequencespecific transcription factors (Nakatani, Y. (2001) Genes Cells 6, 79-86). Expression of PCAF results in increased acetylation of lysine residues (Lys125 and Lys128) within the catalytic cleft of PTEN, a structure essential for phosphatidylinositol 3,4,5-trisphosphate specificity (Lee, J. O., Yang, H., Georgescu, M. M., Di Cristofano, A., Maehama, T., Shi, Y., Dixon, J. E., Pandolfi, P., and Pavletich, N. P. (1999) Cell 99, 323-334). The acetylation of PTEN caused by PCAF expression depends on the presence of growth factors. Reduction of endogenous PCAF activity using shRNA results in a loss of PTEN acetylation in response to growth factors and restores the ability of PTEN to down-regulate phosphatidylinositol 3-kinase signaling and to induce G1 cell cycle arrest. The retention of phosphatidylinositol 3-kinase/AKT signaling and cell cycle regulatory activities of acetylationresistant PTEN K125R and K128R mutants in the presence of enforced PCAF expression suggest a causal relationship. Together, these findings indicate a mechanism of PTEN regulation that forges a link between distinct cancer-relevant pathways central to the control of growth factor signaling and gene expression.

A novel alternatively spliced viral mRNA transcribed in cells infected with human T cell leukemia virus type 1 is mainly responsible for expressing p21X protein

The pX sequence of human T cell leukemia virus type 1 (HTLV‐1) has been thought to be expressed as a doubly spliced mRNA that codes for p40tax, p27rex and p21X. However, we identified a novel alternatively spliced mRNA in the HTLV‐1 infected cells by using reverse transcription followed by the polymerase chain reaction. This mRNA contains only the first and third exons of the doubly spliced mRNA and encodes only p21X. Our data that this mRNA is responsible for expressing p21X exists in most of HTLV‐1 infected cells strongly suggests that p21X may play a crucial role for HTLV‐1 replication.

PTEN - A novel anti-oncogenic function independent of phosphatase activity

The PTEN gene is an important tumor suppressor mutated in a number of cancers. To date, its growth regulatory properties have been intimately linked to its ability to act as a protein and phosphoinositol phosphatase. Inactivation of the enzymatic activity of PTEN is primarily due to direct mutation of its amino-terminal catalytic domain but ~20% of mutations are in the carboxy-terminus, which regulates membrane localization, protein stability, cellular migration and p53 function. We identified a novel protein that interacts with this domain, the v-jun transcriptional target, MSP58. Binding of MSP58 to PTEN results in the suppression of MSP58-mediated transformation. However, this PTEN effect does not require its catalytic activity, suggesting additional mechanisms of PTEN action.

Autoantigen Ku protein is involved in DNA binding proteins which recognize the U5 repressive element of human T-cell leukemia virus type I long terminal repeat

We have identified and analyzed a 27‐nucleotide sequence (U5 repressive element, designated as U5RE) at the U5 region of the human T‐cell leukemia virus type I (HTLV‐I) long terminal repeat (LTR) which is required for HTLV‐I basal transcriptional repression. The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T‐cells in the presence or absence of viral transactivator tax protein. We consistently observed a 2‐to 5‐fold increase in basal promoter activity when sequences between +277 to +306 were deleted. In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (USRE). Using gel mobility shift assays, we have purified a highly enriched fraction that could specifically bind U5RE. This DNA affinity column fraction contained three major detectable proteins on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with silver staining: 110‐, 80‐ and 70‐kDa proteins. The 110‐kDa protein appeared to be a novel DNA‐binding protein whose characteristics are still obscure, while the 70‐ and 80‐kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p70/p80) complex, as demonstrated by amino acid sequencing and immunological analyses. As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with U5RE raises intriguing possibilities for its function in HTLV‐I basal transcriptional repression.

Cloning and sequencing the streptolysin O genes of group C and group G Streptococci

On the basis of the known streptolysin O (SLO) genomic sequence of Streptococcus pyogenes group A, we identified the SLO genes in some strains of group C and group G streptococci by the polymerase chain reaction procedure (PCR). The entire open reading frame region of these genes was cloned and analyzed. Their nucleotide sequence data showed that the defined SLO genes in group C and group G are almost identical to that of group A.