Inactivation of the NF2 tumor suppressor protein merlin in DU145 prostate cancer cells.
BACKGROUND
The neurofibromatosis 2 (NF2) tumor suppressor gene product merlin is an important regulator of contact-dependent cell proliferation. Phosphorylation of merlin at serine 518 (Ser518) by the Rac effector p21-activated kinase (PAK) inactivates merlin's growth suppressing function, and is regulated by cell-culture conditions, including cell density, cell/substrate attachment, and growth factor availability. We examined the regulation of merlin expression and merlin phosphorylation in prostate cancer cells.


METHODS
Phosphorylation of merlin in five prostate cancer cell lines (LNCaP, DU145, PC3, 22RV1, and LAPC-4) was examined by Western blotting using anti-phospho-merlin (Ser518) antibody. The activity of PAK, an upstream regulator of merlin phosphorylation, was measured by Western blotting using phospho-PAK (Ser141) antibody. The effects of various cell-culture conditions on the phosphorylation levels of merlin and PAK were analyzed.


RESULTS
Both merlin expression and phosphorylation were low in LNCaP, PC3, 22RV1, and LAPC-4 prostate cancer cells. In DU145 cells, total and phosphorylated merlin were abundant, but phosphorylation was not inhibited by high cell density, serum withdrawal, the addition of hyaluronic acid or inhibition of CD44 expression, all of which are reported to inhibit merlin phosphorylation in non-neoplastic cells. PAK activation was elevated in DU145 cells and the addition of a PAK-specific inhibitor peptide but not the Rac1-specific inhibitor NSC23766 inhibited both PAK and merlin phosphorylation.


CONCLUSIONS
Merlin is inactivated in DU145 prostate cancer cells by PAK-mediated constitutive phosphorylation, identifying a novel mechanism of merlin inactivation in neoplastic cells.
Horiguchi
Akio
A
Urologic Oncology Research Laboratory, Department of Urology, Weill Cornell Medical College of Cornell University, New York, New York, USA.
Zheng
Rong
R
Shen
Ruoqian
R
Nanus
David M
DM
D015153:Blotting, Western; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D025581:Neurofibromin 2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D054462:p21-Activated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C497483:CD44 protein, human; D004791:Enzyme Inhibitors; D018960:Hyaluronan Receptors; D025581:Neurofibromin 2; D034741:RNA, Small Interfering; D006820:Hyaluronic Acid; D054462:p21-Activated Kinases