Stimulation of Gα q -coupled GPCRs releases PLCβ1 from stress granule–associated proteins, enabling their aggregation.

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.

During adverse environmental conditions, mammalian cells regulate protein production by sequestering the translation machinery in membraneless organelles (i.e. stress granules) whose formation is carefully regulated. In this study, we show a direct connection between G protein signaling and stress granule formation through phospholipase Cβ (PLCβ). In cells, PLCβ localizes to both the cytoplasm and plasma membrane. We find that a major population of cytosolic PLCβ binds to stress granule proteins; specifically, eIF5A and Ago2, whose RNA-induced silencing activity is halted under stress. PLCβ1 is activated by Gαq in response to hormones and neurotransmitters and we find that activation of Gαq shifts the cytosolic population of PLCβ1 to the plasma membrane, releasing stress granule proteins. This release is accompanied by the formation of intracellular particles containing stress granule markers, an increase in the size and number of particles, and a shift of cytosolic RNAs to larger sizes consistent with cessation of transcription. Arrest of protein synthesis is seen when the cytosolic level of PLCβ1 is lowered by siRNA or by osmotic stress, but not cold, heat or oxidative stress causes similar behavior. Our results fit a simple thermodynamic model in which eIF5a and its associated proteins partition into particles after release from PLCβ1 due to Gαq stimulation. Taken together, our studies show a link between Gαq-coupled signals and transcription through stress granule formation.

Cells have developed lineage-specific mechanisms to control proliferation and drive morphologic changes upon differentiation. A hallmark of differentiation is the assembly of signaling molecules that transduce extracellular signals, such as the production of the G protein-regulated enzyme phospholipase Cβ (PLCβ), which generates calcium signals from sensory stimuli. We found that in most cancerous cell lines there is positive correlation between PLCβ1 levels and cell proliferation. In cells of neuronal lineage, however, reducing PLCβ1 levels increases the rate of proliferation. Using a combination of biochemical and biophysical methods, we find that, in the G phase, a cytosolic population of PLCβ1 associates with cyclin-dependent kinase 16 (CDK16), a neuron-specific enzyme that is activated by cyclin Y to inactivate the antioncogenic protein p27. Binding of PLCβ1 directly inhibits CDK16 activity and in turn reduces the ability of cells to enter the S phase. Activation of Gα by carbachol causes movement of PLCβ from the cytosol to the plasma membrane, reducing its association with CDK16. Similarly, the overexpression of activated Gα moves PLCβ1 to the membrane, reverses G arrest, and promotes proliferation, thereby connecting external stimuli with cell proliferation. Our results present a model in which the transient high expression of PLCβ1 that occurs at the onset of differentiation arrests cells in the G phase through its association with CDK16 and allows CDK16 to transition to its postmitotic function of neurite outgrowth and trafficking of synaptic vesicles. The novel role of PLCβ1 in neuronal cell proliferation offers a unique interaction that can be manipulated to guide cells into a neuronal phenotype or to develop therapies for neuroblastomas.-Garwain, O., Valla, K., Scarlata, S. Phospholipase Cβ1 regulates proliferation of neuronal cells.

Phospholipase Cβ1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters. Besides carrying out this plasma membrane function, PLCβ1 has a cytosolic population that helps to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA‐induced silencing (C3PO). Here, we show that down‐regulating PLCβ1 or reducing its cytosolic population by activating Gαq to localize it to the plasma membrane returns differentiated PC12 and SK‐N‐SH cells to an undifferentiated state. In this state, PC12 cells have a spherical morphology, resume proliferation, and express the stem cell transcription factors nanog and Oct4. Similar changes are seen when C3PO is down‐regulated. This return to a stem‐like state is accompanied by shifts in multiple miR populations. Surprisingly, de‐differentiation can be induced by extended stimulation of Gαq where cells return to a spherical morphology and levels of specific miRs return to their undifferentiated values. In complementary studies, we followed the real‐time hydrolysis of a fluorescent‐tagged miR in cells where PLCβ1 or C3PO were down‐regulated in PC12 cells and find substantial differences in miR processing in the undifferentiated and differentiated states. Taken together, our studies suggest that PLCβ1, through its ability to regulate C3PO and endogenous miR populations, mediates the differentiation of two types of cultured neuronal cells.