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. 2006 Oct 16;580(24):5779-84.
doi: 10.1016/j.febslet.2006.09.035. Epub 2006 Sep 27.

PHI-1 interacts with the catalytic subunit of myosin light chain phosphatase to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force in avian smooth muscle

Amr El-Toukhy  1 , Allison M Given, Ozgur Ogut, Frank V Brozovich
Affiliations

PHI-1 interacts with the catalytic subunit of myosin light chain phosphatase to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force in avian smooth muscle

Amr El-Toukhy et al. FEBS Lett. .

Abstract

In avian smooth muscles, GTPgammaS produces a Rho kinase mediated increase in PHI-1 phosphorylation and force, but whether this correlation is causal is unknown. We examined the effect of phosphorylated PHI-1 (P-PHI-1) on force and myosin light chain (MLC(20)) phosphorylation at a constant [Ca(2+)]. P-PHI-1, but not PHI-1, increased MLC(20) phosphorylation and force, and phosphorylation of PHI-1 increased the interaction of PHI-1 with PP1c. Microcystin induced a dose-dependent reduction in the binding of PHI-1 to PP1c. These results suggest PHI-1 inhibits myosin light chain phosphatase by interacting with the active site of PP1c to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force.

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Figures

Figure 1
Figure 1. Rho kinase phosphorylates PHI-1
Purified PHI-1 was phosphorylated by Rho kinase (see text for details). Lane 1 shows the western blot of the purified protein, and lane two after Rho kinase treatment. Western blots were probed with anti-PHI-1 and antiphospho-PHI-1 antibodies. The Thr57 phospho-specific antibody only recognized the protein after phosphorylation.
Figure 2
Figure 2. Rho signaling is intact after Triton skinning
The expression of RhoA and Rho kinase was determined in intact smooth muscle and Triton skinned smooth muscle. As demonstrated, both RhoA and Rho kinase are retained following skinning of the smooth muscle.
Figure 3
Figure 3. P-PHI-1 leads to Ca2+ sensitization
(A) A skinned gizzard strip was placed in relaxing solution (pCa9) and then at the first transferred to pCa6.2. P-PHI-1 (3 μg/ml) was added to the pCa6.2 and the preparation was then transferred to pCa4 solution (without PHI-1). The pCa solutions changes and the addition of P-PHI-1 are indicated below the data trace. (B) A skinned gizzard strip was placed in relaxing solution (pCa9) and then transferred to pCa6.2. PHI-1 (3 μg/ml) and then P-PHI-1 (1st, 3 μg/ml and then 6 μg/ml) was added to the pCa6.2 solution. The fiber was then transferred to pCa4 solution (without PHI-1 or P-PHI-1) and then to pCa9 solution (without PHI-1 or P-PHI-1). The pCa solutions changes and the addition of PHI-1 and P-PHI-1 are indicated below the data trace. As is demonstrated, the addition of P-PHI-1, but not PHI-1, resulted in a significant increase in force.
Figure 4
Figure 4. P-PHI-1 increases MLC20 phosphorylation
MLC20 phosphorylation was determined in skinned gizzard strips (n=4 for each condition) at pCa6.2 alone, pCa6.2 with PHI-1, pCa6.2 with P-PHI-1, as well as pCa4 (see Table II).
Figure 5
Figure 5. P-PHI-1 binds to the active site of PP1c
(A) Representative immunoprecipitation with anti-PP1c antibody and Western blot for PHI-1, which demonstrate the interaction of PP1c and PHI-1. Immunoprecipitation was also performed following the addition of GTPγS and microcytin to the tissue lysates. (B) Western blots were analyzed using densitometry, and the bar graph summarizing the results (n=3–6). The immunoprecipitations demonstrate an interaction between PP1c and PHI-1, which increases with phosphorylation of PHI-1. The addition of Microcystin LR results in a dose dependent decrease in the interaction of PP1c and PHI-1.
Supplemental Figure
Supplemental Figure. GTPγS does not affect immunoprecipitation of PP1c
Western blot for PP1c in gizzard lysate (lane 1). Endogenous PP1c was immunoprecipitated from gizzard lysates in the absence (lane 2) and presence (lane 3) of GTPγS. Similar amounts of PP1c were detected in the immunoprecipitation with and without GTPγS.
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