TBC1D16 is a Rab4A GTPase activating protein that regulates receptor recycling and EGF receptor signaling.

Rab4A is a master regulator of receptor recycling from endocytic compartments to the plasma membrane. The protein TBC1D16 is up-regulated in melanoma, and TBC1D16-overexpressing melanoma cells are dependent on TBC1D16. We show here that TBC1D16 enhances the intrinsic rate of GTP hydrolysis by Rab4A. TBC1D16 is both cytosolic and membrane associated; the membrane-associated pool colocalizes with transferrin and EGF receptors (EGFRs) and early endosome antigen 1, but not with LAMP1 protein. Expression of two TBC1D16 isoforms, but not the inactive R494A mutant, reduces transferrin receptor recycling but has no effect on transferrin receptor internalization. Expression of TBC1D16 alters GFP-Rab4A membrane localization. In HeLa cells, overexpression of TBC1D16 enhances EGF-stimulated EGFR degradation, concomitant with decreased EGFR levels and signaling. Thus, TBC1D16 is a GTPase activating protein for Rab4A that regulates transferrin receptor recycling and EGFR trafficking and signaling.

Upregulation of the Catalytic Telomerase Subunit by the Transcription Factor ER81 and Oncogenic HER2/Neu, Ras, or Raf.

One hallmark of tumor formation is the transcriptional upregulation of human telomerase reverse transcriptase, hTERT, and the resultant induction of telomerase activity. However, little is presently understood about how hTERT is differentially activated in tumor cells versus normal somatic cells. Specifically, it is unclear if oncoproteins can directly elicit hTERT expression. To this end, we now show that three oncoproteins, HER2/Neu, Ras, and Raf, stimulate hTERT promoter activity via the ETS transcription factor ER81 and ERK mitogen-activated protein (MAP) kinases. Mutating ER81 binding sites in the hTERT promoter or suppression of ERK MAP kinase-dependent phosphorylation of ER81 rendered the hTERT promoter unresponsive to HER2/Neu. Further, expression of dominant-negative ER81 or inhibition of HER2/Neu significantly attenuated telomerase activity in HER2/Neu-overexpressing SKBR3 breast cancer cells. Moreover, HER2/Neu, Ras, and Raf collaborated with ER81 to enhance endogenous hTERT gene transcription and telomerase activity in hTERT-negative, nonimmortalized BJ foreskin fibroblasts. Accordingly, hTERT expression was increased in HER2/Neu-positive breast tumors and breast tumor cell lines relative to their HER2/Neu-negative counterparts. Collectively, our data elucidated a mechanism whereby three prominent oncoproteins, HER2/Neu, Ras, and Raf, may facilitate tumor formation by inducing hTERT expression in nonimmortalized cells via the transcription factor ER81.

Regulation of telomerase reverse transcriptase gene activity by upstream stimulatory factor.

Upregulation of human telomerase reverse transcriptase (hTERT) transcription accounts for the immortalization of greater than 85% of all human tumor cells. However, the mechanism whereby hTERT expression is activated remains unresolved. Specifically, recent data challenging the role of Myc/Max in E-box-dependent activation of hTERT expression suggests that other E-box-binding proteins regulate hTERT transcription. Indeed, we now demonstrate that two such proteins, upstream stimulatory factor (USF) 1 and 2, readily associate with two E-boxes in the hTERT promoter in vitro and in vivo primarily as heterodimers, whereas Myc/Max does not. The avid binding of USF1/2 heterodimers to these E-boxes occurs in both hTERT-positive and -negative cells. In contrast, USF1/2 activates the hTERT promoter exclusively in hTERT-positive cells in a manner that is enhanced by the coactivator p300 and attenuated upon inhibiting p38-MAP kinase, a known modulator of USF activity. Collectively, our data indicate that USF binding to the hTERT promoter may be transcriptionally neutral, or even repressive, in nonimmortalized hTERT-negative somatic cells, but stimulatory in hTERT-positive cells where USF1/2 contributes to the acquisition and maintenance of immortality.