Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT)-3 by thyroid oncogenic kinase RET/PTC.
BACKGROUND
RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although it has been established that RET/PTC kinase plays a crucial role in intracellular signaling pathways that regulate cellular transformation, growth, and proliferation in thyroid epithelial cells, the upstream signaling that leads to the activation of RET/PTC is largely unknown. Based on the observation of high levels of PLD expression in human papillary thyroid cancer tissues, we investigated whether PLD plays a role in the regulating the RET/PTC-induced STAT3 activation.


METHODS
Cancer tissue samples were obtained from papillary thyroid cancer patients (n = 6). The expression level of PLD was examined using immunohistochemistry and western blotting. Direct interaction between RET/PTC and PLD was analyzed by co-immunoprecipitation assay. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol, the product of PLD-mediated transphosphatidylation, in the presence of n-butanol. The transcriptional activity of STAT3 was assessed by m67 luciferase reporter assay.


RESULTS
In human papillary thyroid cancer, the expression levels of PLD2 protein were higher than those in the corresponding paired normal tissues. PLD and RET/PTC could be co-immunoprecipitated from cells where each protein was over-expressed. In addition, the activation of PLD by pervanadate triggered phosphorylation of tyrosine 705 residue on STAT-3, and its phosphorylation was dramatically higher in TPC-1 cells (from papillary carcinoma) that have an endogenous RET/PTC1 than in ARO cells (from anaplastic carcinoma) without alteration of total STAT-3 expression. Moreover, the RET/PTC-mediated transcriptional activation of STAT-3 was synergistically increased by over-expression of PLD, whereas the PLD activity as a lipid hydrolyzing enzyme was not affected by RET/PTC.


CONCLUSIONS
These findings led us to suggest that the PLD synergistically functions to activate the STAT3 signaling by interacting directly with the thyroid oncogenic kinase RET/PTC.
Kim
Young-Rae
YR
Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon, 301-131, South Korea. young7403@hanmail.net
Byun
Hee Sun
HS
Won
Minho
M
Park
Kyeong Ah
KA
Kim
Jin Man
JM
Choi
Byung Lyul
BL
Lee
Hyunji
H
Hong
Jang Hee
JH
Park
Jongsun
J
Seok
Jeong Ho
JH
Kim
Dong Wook
DW
Shong
Minho
M
Park
Seung-Kiel
SK
Hur
Gang Min
GM
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002291:Carcinoma, Papillary / Q000201:enzymology* / Q000473:pathology; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D013964:Thyroid Neoplasms / Q000201:enzymology* / Q000473:pathology; D015533:Transcriptional Activation
D050796:STAT3 Transcription Factor; D051096:Proto-Oncogene Proteins c-ret; D010739:Phospholipase D