Korean J Physiol Pharmacol.  2018 Jan;22(1):91-99. 10.4196/kjpp.2018.22.1.91.

Functional identification of protein phosphatase 1-binding consensus residues in NBCe1-B

• Lee KP
• Kim HJ
• Yang D

• KMID: 2398559
• DOI: http://doi.org/10.4196/kjpp.2018.22.1.91

Abstract

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, ⁹²²FMDRLK⁹²â· , in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922-927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO₃⁻ transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO₃⁻ secretion in several types of epithelia.

Keyword

HCO₃⁻ secretion; IRBIT; NBCe1-B; Protein phosphatase 1; SPAK; WNK

MeSH Terms

Amino Acids
Catalytic Domain
Consensus*
Hydrogen-Ion Concentration
Immunoprecipitation
Mutagenesis, Site-Directed
Protein Phosphatase 1
Signal Transduction
Amino Acids
Protein Phosphatase 1

Figure

• 

  Fig. 1 Conservation of the PP1-binding consensus residues in NBCe1-B.(A) Sequence alignment analysis: the C-terminal part of the human NBCe1-B (Q9Y6R1.1) against the orthologous transporters of Mus musculus (O88343.2), Rattus norvegicus (Q9JI66.1), Bos taurus (NP_777030.1), and Trichosurus vulpecula (AEQ33587.1). (B) Amino acids (downward-pointing arrows) are critical in binding to PP1, and substituted by site-directed mutagenesis. (A, B) The PP1-binding consensus residues are indicated using bold bars.

• 

  Fig. 2 PP1 interacts with the PP1-binding consensus residues in NBCe1-B.(A) Co-immunoprecipitation of NBCe1-B with PP1 in HEK293T cells transfected with PP1 co-expressing NBCe1-B, 3-point mutant, and PP1-binding consensus residue deletion mutant. The cells were lysed with RIPA buffer. The extract was immunoprecipitated with anti-GFP or anti-PP1. (B) HEK293T cells transfected with 3-point mutant or PP1-binding consensus residue deletion mutant showing a significant decrease of NBCe1-B activity via association with PP1 (28.9±9.3% and 18.2±9.1%; n=3; *p<0.05). *Significance was compared against the wild type value.

• 

  Fig. 3 Role of PP1-binding consensus residues in NBCe1-B.(A–C) Measurement of NBCe1-B activity in HeLa cells transfected with wild type, 3-point mutant, and PP1-binding consensus residue deletion mutant by monitoring intracellular pH. (D) HeLa cells transfected with 3-point mutant or PP1-binding consensus residue deletion mutant showing a significant decrease of NBCe1-B activity (45.1±6.5% and 35.7±4.7%; *p<0.05). *Significance was compared against the wild type value. Number of experiments for each condition are shown below the X-axis. Recovery, the rates of Na+-dependent changes in pHi (Δ pHi (recovery)/Δ pHi (total)); Slope, the slopes of the first derivatives of pHi increase in HCO3−-buffered media containing 140 mM of Na+(Δ pHi/min).

• 

  Fig. 4 PP1 stimulates NBCe1-B activity via PP1-binding consensus residues in NBCe1-B.(A–D) Measurement of NBCe1-B activity in HeLa cells transfected with wild type, 3-point mutant, and PP1-binding consensus residue deletion mutant co-expressing PP1 by monitoring intracellular pH. (E) NBCe1-B with PP1 shows a significant increase in NBCe1-B activity, but 3-point mutant and PP1-binding consensus residue deletion mutant co-expressing PP1 inhibit NBCe1-B activity (155.3±5.9%, 53.1±6.4% and 47.2±7.3%; *p<0.05). *Significance was compared against the wild type+empty vector value. Number of experiments for each condition are shown below the X-axis. Recovery, the rates of Na+-dependent changes in pHi (Δ pHi (recovery)/Δ pHi (total)); Slope, the slopes of the first derivatives of pHi increase in HCO3−-buffered media containing 140 mM of Na+(Δ pHi/min).

• 

  Fig. 5 NBCe1-B surface expression is associated with PP1.(A) Biotinylation of NBCe1-B with or without PP1 in HEK293T cells transfected with or without PP1 co-expressing NBCe1-B, 3-point mutant, and PP1-binding consensus residue deletion mutant. Cells were incubated with biotin. Biotinylated NBCe1-B was isolated and recovered. Input corresponds to 5% of the total NBCe1-B used for the avidin pull-down step. (B) NBCe1-B with PP1 shows an approximate 2-fold increase of NBCe1-B surface expression level (210.5±16.9%). However, 3-point mutant and PP1-binding consensus residue deletion mutant with or without PP1 significantly inhibit NBCe1-B surface expression level (29.5±12.6%, 37.2±11.8%, 15.3±4.2% and 12.6±4.3%, respectively). Significant differences (*p<0.05) between wild type and each experimental group were statistically determined by one-way ANOVA.

• 

  Fig. 6 Regulation of NBCe1-B via the IRBIT/PP1 and WNK/SPAK pathways.The WNK/SPAK pathway keeps the epithelial ductal cells in a steady state by decreasing the surface expression of NBCe1-B. The WNKs directly bind to SPAK, which phosphorylates NBCe1-B. After cell stimulation, IRBIT inhibits the WNK/SPAK pathway and activates NBCe1-B. IRBIT interacts with PP1 and PP1 is recruited to NBCe1-B by IRBIT. Thus, PP1 specifically docks with the PP1-binding consensus residue in NBCe1-B and dephosphorylates it, thereby restoring its surface expression.

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