Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca2+ sensitization force of smooth muscle

Figures

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  Fig. 1.. Structure of CPI-17 and the recombinant proteins used. (A) Cluster Omega program (EMBL-EBI) was used for alignment and phylogenic tree generation of amino acid sequences of CPI-17 (PPP1R14A). Blue lines indicate the unstructured regions at N-and C-termini. An asterisk indicates positions fully conserved, a colon and period indicate positions in which chemical properties of residues are conserved. (B) Schematic illustration of recombinant CPI-17 proteins used for the assays. Box indicates the central inhibitory domain (PHIN domain). Circles indicate Ser12, Thr38 and Ser128, whose phosphorylation occurs in cells.

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  Fig. 2.. Force production of permeabilized strips induced by recombinant CPI-17 proteins. (A) Representative relative force trace of the permeabilized smooth muscle strips. Permeabilized smooth muscle strips was doped with recombinant CPI-17 (10 µM) in the G1 solution, and then stimulated with the pCa6.3 solution including 3 µM PDBu. After the force reached to plateau, the maximum extent of force was induced with the pCa4.5 solution including 1 µM MCLR. Bar indicates 10 min. (B) Representative force traces in the presence of WT (blue), dN (green), dC (orange), dNC (black) and T38A (gray). Red line indicates force trace without recombinant CPI-17. (C) Dot plot of the Ca2+ sensitization force induced by recombinant CPI-17. Mean values of relative extent of force development are shown in horizontal lines. * indicates p<0.05 by Dunnett’s test vs. WT.

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  Fig. 3.. Phosphorylation of recombinant CPI-17 by kinases in smooth muscle extracts. (A) Recombinant CPI-17 proteins were phosphorylated in the presence of fundus smooth muscle extracts and subjected to phospho-dot blot assay. Densitometric data of each dot were normalized against the values of the WT protein. Mean values of relative extent of phosphorylation are shown in horizontal lines. * indicates p<0.05 by Dunnett’s test vs. WT. (B) Phosphorylation of each protein was conducted in the presence of 3 µM GFx and 1 µM H1152. * indicates p<0.05 by Dunnett’s test vs. control without antagonist (Ctl).

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  Fig. 4.. Binding of CPI-17 with myosin phosphatase in smooth muscle extracts. Pulldown assay with recombinant CPI-17 proteins were conducted using smooth muscle cell extracts as a source of the myosin phosphatase. Myosin phosphatase bound to the resins coupled with CPI-17 proteins was detected by immunoblotting using anti-PP1 delta antibody (A). Each densitometric value of the bound PP1 delta was normalized by that of the extent CPI-17 on the beads. Mean values of relative binding of PP1 are shown in horizontal lines (B). Ext; an input sample subjected to the pulldown assay, BLK; blank beads without protein. Numbers indicate molecular weight in kDa. * indicates p<0.05 by Dunnett’s test vs. WT.

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  Fig. 5.. Homologs of the CPI-17 family in avian species. BLASTP searches for avian homologs of CPI-17, PHI-1, KEPI and GBPI in GenBank were conducted using each human sequence as a bait. For CPI-17, sequences with over 80% identity and E value of >6e-52 were considered as avian CPI-17 homologs that include small tree finch (Camarhynchus parvenus), lance-tailed manakin (Chiroxiphia lanceolate) bald eagle (Haliaeetus leucocephalus). Phylogenic tree (top) indicates similarity of four CPI-17 family members of small tree finch. Venn diagram (bottom) indicates distribution of the homologs of avian species.