Phospholipase Cβ-TRAX Association Is Required for PC12 Cell Differentiation.
When treated with nerve growth factor, PC12 cells will differentiate over the course of several days. Here, we have followed changes during differentiation in the cellular levels of phosphoinositide-specific phospholipase Cβ (PLCβ) and its activator, Gαq, which together mediate Ca2+release. We also followed changes in the level of the novel PLCβ binding partner TRAX (translin-associated factor X), which promotes RNA-induced gene silencing. We find that the level of PLCβ increases 4-fold within 24 h, whereas Gαqincreases only 1.4-fold, and this increase occurs ∼24 h later than PLCβ. Alternately, the level of TRAX remains constant over the 72 h tested. When PLCβ1 or TRAX is down-regulated, differentiation does not occur. The impact of PLCβ on differentiation appears independent of Gαqas down-regulating Gαqat constant PLCβ does not affect differentiation. Förster resonance energy transfer studies after PLCβ association with its partners indicate that PLCβ induced soon after nerve growth factor treatment associates with TRAX rather than GαqFunctional measurements of Ca2+signals to assess the activity of PLCβ-Gαqcomplexes and measurements of the reversal of siRNA(GAPDH) to assess the activity of PLCβ-TRAX complexes additionally suggest that the newly synthesized PLCβ associates with TRAX to impact RNA-induced silencing. Taken together, our studies show that PLCβ, through its ability to bind TRAX and reverse RNA silencing of specific genes, plays a key role in switching PC12 cells to their differentiated state.
From the Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609.
Osama
O
Garwain
From the Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609 sfscarlata@wpi.edu.
Suzanne
S
Scarlata
D000818:Animals; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells / Q000166:cytology / Q000201:enzymology*; D054799:Phospholipase C beta / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats
D004268:DNA-Binding Proteins; C498307:Tsnax protein, rat; D054799:Phospholipase C beta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11
G protein; Phospholipase C; calcium intracellular release; cell differentiation; fluorescence resonance energy transfer (FRET)