Phospholipase Cβ-TRAX Association Is Required for PC12 Cell Differentiation

Abstract

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, Gα, which together mediate Ca 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 Gα increases 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 Gα as down-regulating Gα at 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 Gα Functional measurements of Ca signals to assess the activity of PLCβ-Gα complexes 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.

Phospholipase Cb1 regulates proliferation of neuronal cells

“…When PC12 cells are treated with nerve growth factor, proliferation ceases and neurites form (31, 32). Our previous studies show that the levels of PLCβ1 increase dramatically in the first 24 h, long before the levels of Gα q rise (1). Thus, our studies give rise to a model in which the bolus of PLCβ1 produced at the onset of differentiation binds to CDK16 to prevent entry into the S phase.…”

“…Western blot was carried out as previously described (1) using the following primary antibodies: PLCβ1 (D‐8; Santa Cruz Biotechnology, Dallas, TX, USA), Gα q (E‐17; Santa Cruz Biotechnology), β‐actin (Abcam, Cambridge, United Kingdom), PO4‐Ser10‐p27 Kip1 (Abcam), and glyceraldehyde 3‐phosphate dehydrogenase (Abcam).…”

“…In previous work, we found that increased PLCβ1 levels as well a transient interaction with C3PO were necessary for cell differentiation (1). In carrying out these studies, however, we noted that PLCβ1 levels had a direct effect on cell proliferation.…”

“…The transition of cells from a rapidly dividing state to a differentiated state requires the arrest of proliferation and the initiation of mechanisms that will define the cell's specific morphology and function. In the neuronal cell line PC12, differentiation is accompanied by an increased amount of the phospholipase Cb1 (PLCb1) (1). PLCb1 is highly expressed in neuronal cells and is associated with neuronal development (2)(3)(4)(5)(6).…”

Simulation of the Gαq /Phospholipase Cβ1 Signaling Pathway Returns Differentiated PC12 Cells to a Stem-like State

“…However, C3PO competes for the same site on PLCβ as Gαq and increasing its cellular levels quenches calcium signaling mediated through Gαq receptors [6][7][8]. It is notable that increasing the level of activated Gαq by transfection or stimulation, will result in dissociation of PLCβ from C3PO and allow C3PO to promote RNA-induced silencing [7,8].In previous studies, we found that cytosolic PLCβ1 is required for differentiation of PC12 cells [9]. PC12 cells are a widely used model for neuronal cell differentiation [10].…”