p116^Rip Targets Myosin Phosphatase to the Actin Cytoskeleton and Is Essential for RhoA/ROCK-regulated Neuritogenesis

Abstract

Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCKactomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116Rip , whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116Rip , through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116Rip inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116Rip is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.

An association between NUAK2 and MRIP reveals a novel mechanism for regulation of actin stress fibers

“…MYPT1 was not efficiently expressed in our hands possibly owing to toxicity, whereas the functionally closely related (Mulder et al, 2004;Zagorska et al, 2010) MBS85 cDNA (PPP1R12C) was readily detectable. GFP-MBS85 alone localized partially along actin fibers, the levels of which were significantly decreased ( Fig.…”

“…The results suggest that the association of NUAK2 and MRIP is through the MRIP coiled-coiled region, including the part crucial for MYPT1 interaction (Fig. 1A) (Mulder et al, 2004;Surks et al, 2003).…”

“…1A), suggesting a direct mechanism for targeting of NUAK2 to actin stress fibers. An alternative indirect model is suggested by interactions between, on the one hand MRIP and MYPT1 (Mulder et al, 2004;Surks et al, 2003) and, on the other, between MYPT1 and NUAK2 (Yamamoto et al, 2008). To investigate this, the distribution of GST-NUAK2 was examined following small interfering RNA (siRNA)-mediated downregulation of either MRIP or MYPT1, where expression levels of ectopically expressed and downregulated proteins were analyzed by western blotting (Fig.…”

“…On actin stress fibers, MYPT1 (PPP1R12A) and its close family member MBS85 (PPP1R12C) also interact with the actin-filamentbinding protein MRIP (also known as MPRIP and p116Rip) (Mulder et al, 2004;Surks et al, 2003). Several observations suggest that this interaction participates in the recruitment of MLCP to dephosphorylate MLC on actomyosin fibers: (1) MRIP directly associates with actin filaments through an N-terminal domain containing a pleckstrin homology (PH) domain and an acidic stretch (Mulder et al, 2003) and with MYPT through its C-terminal coil-coiled regions (Mulder et al, 2004;Surks et al, 2003) (shown schematically in Fig.…”

“…Several observations suggest that this interaction participates in the recruitment of MLCP to dephosphorylate MLC on actomyosin fibers: (1) MRIP directly associates with actin filaments through an N-terminal domain containing a pleckstrin homology (PH) domain and an acidic stretch (Mulder et al, 2003) and with MYPT through its C-terminal coil-coiled regions (Mulder et al, 2004;Surks et al, 2003) (shown schematically in Fig. 1A), (2) enhanced MRIP expression in cells leads to disruption of actin stress fibers (Mulder et al, 2003), (3) MRIP downregulation increases actin stress bundles and MLC phosphorylation, and shifts MYPT1 from a particulate to a soluble fraction (Koga and Ikebe, 2005;Surks et al, 2005), and (4) actin stress fibers in cells where MRIP (Surks et al, 2005) or MYPT (Xia et al, 2005) is downregulated are not disassembled following inhibition of ROCK by Y27632, implying that MLCP requires the association of MYPT and MRIP for efficient dephosphorylation of MLC.…”