Abstract
Transforming growth factor-β membrane associated protein (TIMAP) is an endothelial cell (EC)–predominant PP1 regulatory subunit and a member of the myosin phosphatase target (MYPT) protein family. The MYPTs preferentially bind the catalytic protein phosphatase 1 subunit PP1cβ, forming myosin phosphatase holoenzymes. We investigated whether TIMAP/PP1cβ could also function as a myosin phosphatase. Endogenous PP1cβ, myosin light chain 2 (MLC2), and myosin IIA heavy chain coimmunoprecipitated from EC lysates with endogenous TIMAP, and endogenous MLC2 colocalized with TIMAP in EC projections. Purified recombinant GST-TIMAP interacted directly with purified recombinant His-MLC2. However, TIMAP overexpression in EC enhanced MLC2 phosphorylation, an effect not observed with a TIMAP mutant that does not bind PP1cβ. Conversely, MLC2 phosphorylation was reduced in lung lysates from TIMAP-deficient mice and upon silencing of endogenous TIMAP expression in ECs. Ectopically expressed TIMAP slowed the rate of MLC2 dephosphorylation, an effect requiring TIMAP–PP1cβ interaction. The association of MYPT1 with PP1cβ was profoundly reduced in the presence of excess TIMAP, leading to proteasomal MYPT1 degradation. In the absence of TIMAP, MYPT1-associated PP1cβ readily bound immobilized microcystin-LR, an active-site inhibitor of PP1c. By contrast, TIMAP-associated PP1cβ did not interact with microcystin-LR, indicating that the active site of PP1cβ is blocked when it is bound to TIMAP. Thus, TIMAP inhibits myosin phosphatase activity in ECs by competing with MYPT1 for PP1cβ and blocking the PP1cβ active site.
Highlights
GFP-TIMAPWT and GFP-TIMAPV64A/F66A expression was observed throughout the cells, whereas GFP alone was nuclear in GFP-vector–transduced cells. pSer19 MLC2 (pMLC2) immunofluorescence was markedly augmented in endothelial cells (ECs) expressing GFP-TIMAPWT but not in ECs expressing GFPTIMAPV64A/F66A
We investigated whether TIMAP/PP1 catalytic (PP1c) acts as a functional myosin phosphatase in living ECs because TIMAP is an EC-predominant MYPT1 family member [22] that supports myosin-dependent EC processes like angiogenesis [30] and maintenance of pulmonary EC barrier integrity [34]
TIMAP partially colocalizes and directly interacts with myosin light chain 2 (MLC2), it inhibits MLC2 dephosphorylation in ECs by competing for PP1c with MYPT1, leading to MYPT1 degradation (Fig. 6)
Summary
We first determined whether TIMAP interacts with its putative substrate, MLC2 (Fig. 1). Contrary to previous findings that TIMAP/PP1c can dephosphorylate pMLC2 in vitro, MLC2 phosphorylation was much greater in ECs overexpressing GFP-WT TIMAP (GFP-TIMAPWT) compared with GFP-vector–transduced ECs (Fig. 2A). This effect of exogenously expressed TIMAP depended on the multiplicity of infection (m.o.i.) and was not observed with expression of GFPTIMAPV64A/F66A, a TIMAP mutant that does not bind PP1c [18] (Fig. 2, B and C). Expression of GFP-TIMAPS333A/S337A, which cannot be phosphorylated by PKA/GSK3 and binds PP1c with high affinity [18], significantly raised PP1c and pMLC2 levels and reduced MYPT1 abundance, effects that were m.o.i.-dependent. TIMAP Ser333/Ser337 phosphorylation does not mitigate these effects
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