Phospholipase C‐η2 is required for retinoic acid‐stimulated neurite growth

Phospholipase C‐η2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCη2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca²⁺ signals by stimulating further Ca²⁺ release from Ins(1,4,5)P₃‐sensitive stores. The role of PLCη2 in neuritogenesis was assessed during retinoic acid (RA)‐induced Neuro2A cell differentiation. PLCη2 expression increased two‐fold during a 4‐day differentiation period. Stable expression of PLCη2‐targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA‐treatment. Furthermore, RA response element activation was perturbed by PLCη2 knockdown. Using a bacterial two‐hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCη2. Immunostaining of PLCη2 revealed significant co‐localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA‐induced phosphorylation of LIMK1 and cAMP‐responsive element‐binding protein was reduced in PLCη2 knock‐down cells. The phosphoinositide‐binding properties of the PLCη2 PH domain, assessed using a FRET‐based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P₃. Immunostaining of PLCη2 together with PtdIns(3,4,5)P₃ in the Neuro2A cells revealed a high degree of co‐localization, indicating that PtdIns(3,4,5)P₃ levels in cellular compartments are likely to be important for the spatial control of PLCη2 signaling.