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

Abstract

Phospholipase C-g2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/ maturation. PLCg2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca 2+ signals by stimulating further Ca 2+ release from Ins(1,4,5)P 3 -sensitive stores. The role of PLCg2 in neuritogenesis was assessed during retinoic acid (RA)-induced Neuro2A cell differentiation. PLCg2 expression increased two-fold during a 4-day differentiation period. Stable expression of PLCg2-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 PLCg2 knockdown. Using a bacterial two-hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCg2. Immunostaining of PLCg2 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 PLCg2 knock-down cells. The phosphoinositide-binding properties of the PLCg2 PH domain, assessed using a FRETbased assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P 3 . Immunostaining of PLCg2 together with PtdIns(3,4,5)P 3 in the Neuro2A cells revealed a high degree of co-localization, indicating that PtdIns(3,4,5)P 3 levels in cellular compartments are likely to be important for the spatial control of PLCg2 signaling.

Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth

“…In accordance, deletion of the chromosomal region 1p36.32 in which PLCη2 is located, leads to mental retardation (Fitzgibbon et al 2008 ; Lo Vasco 2011 ). Previously, using a targeted knockdown approach, we found PLCη2 to be essential for retinoic acid-induced neurite growth in Neuro2A cells (Popovics et al 2013 ). Despite this, the precise mechanism by which PLCη2 facilitates neurite growth in these cells is not known.…”

“…Despite this, the precise mechanism by which PLCη2 facilitates neurite growth in these cells is not known. In our previous study, through use of a bacterial two-hybrid assay we identified Lim-domain kinase-1 (LIMK-1) as a putative C-terminal interaction partner of PLCη2 (Popovics et al 2013 ). LIMK-1 acts primarily downstream of Rho GTPases to phosphorylate and inactivate cofilin family proteins (cofilin1, cofilin2 and destrin), which promote actin depolymerization and the severing of actin filaments during neurite outgrowth (Arber et al 1998 ; Yang et al 1998 ; Endo et al 2007 ).…”

Phospholipase Cη2 Activation Redirects Vesicle Trafficking by Regulating F-actin

“…6 , A and B ). Because PLCη2 was reported to localize to the plasma membrane by binding to PI(4,5)P 2 via its PH domain ( 41 , 48 ), the observed filamentous distribution of EGFP-PLCη2 ( Fig. 6 A ) could result from binding to PI(4,5)P 2 -rich membrane domains that co-localize along F-actin filaments.…”

A Canonical EF‐Loop Directs Ca2+‐Sensitivity in Phospholipase C‐η2

“…Both catalyze hydrolysis of PIP 2 (Nakahara et al, 2005;Hwang et al, 2005;Zhou et al, 2005), which suggests that these enzymes, like other PIP 2 -specific PLCs, are involved in the production of DAG and IP 3 . The cellular function of these enzymes is not entirely clear, although a recent study highlighted a potential role for PLCη2 in regulating neurite growth (Popovics et al, 2013). It has become apparent that both PLCη enzymes are activated by intracellular Ca 2+ mobilization (Kim et al, 2011;Popovics et al, 2011), and are sensitive to changes in [Ca 2+ ] i within the physiological range (Nakahara et al, 2005;Hwang et al, 2005).…”