Korean J Physiol Pharmacol.  2016 Jul;20(4):433-440. 10.4196/kjpp.2016.20.4.433.

Ahcyl2 upregulates NBCe1-B via multiple serine residues of the PEST domain-mediated association

Affiliations
  • 1Department of Laboratory Medicine, Gachon University Gil Hospital, Incheon 21565, Korea.
  • 2Department of Physiology, College of Medicine, Gachon University, Incheon 21936, Korea. dkyang@gachon.ac.kr

Abstract

Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.

Keyword

AHCYL2; HCO(3)- secretion; IRBIT; NBCe1-B

MeSH Terms

Animals
Carrier Proteins
Hydrogen-Ion Concentration
Mice
Mutagenesis, Site-Directed
Phosphorylation
S-Adenosylhomocysteine
Serine*
Carrier Proteins
S-Adenosylhomocysteine
Serine

Figure

  • Fig. 1 Sequence alignment of mIRBIT and Ahcyl2.Amino acids essential for interaction with NBCe1-B, as determined by performing site-directed and deletion mutagenesis, are indicated using upward-pointing arrows and bold bars. The PEST and C-C domains are shown by using bold bars.

  • Fig. 2 Ahcyl2 interacts with specific domains and amino acids of NBCe1-B.(A and C) HEK293T cells were transfected with GFP-NBCe1-B and an empty vector or vectors expressing FLAG-Ahcyl2, FLAG-point, and FLAG-deletion mutants. Interaction of Ahcyl2 with GFP–NBCe1-B was determined by performing coimmunoprecipitation assay. NBCe1-B was detected using anti-GFP antibody, and Ahcyl2 was detected using anti-FLAG antibody. (B and D) Densitometry analysis of efficiency of association of Ahcyl2 with NBce1-B expressed as amount of Ahcyl2 co-precipitates divided by the total amount of Ahcyl2 input [Ahcyl2-Blot/Ahcyl2-Input] (for comparison, results were normalized to 100% for Ahcyl2). (*p<0.01 versus Wild Type, n=3).

  • Fig. 3 Ahcyl2 stimulates NBCe1-B activity.(A~C) HeLa cells were transfected with GFP-NBCe1-B and an empty vector or vectors expressing mIRBIT or Ahcyl2. (D) NBCe1-B activity in transfected HeLa cells was determined by measuring Na+-dependent recovery of acid load in the presence of 10 µM EIPA and normalized relative to control. In all, 9~10 experiments were performed for each condition. Numbers below the labels on the x-axis indicate the frequency of experiments. *p<0.01 versus NBCe1-B.

  • Fig. 4 Role of Ahcyl2 C-C domains in NBCe1-B activation.(A~G) HeLa cells were transfected with GFP-NBCe1-B and an empty vector or vectors expressing Ahcyl2 or its point or C-C domain deletion mutant, (H) NBCe1-B activity was measured and normalized relative to control. Bars indicate mean±SEM of 7~10 separate experiments. *p<0.01 versus NBCe1-B. Numbers below the labels on the x-axis indicate the frequency of experiments.

  • Fig. 5 Role of Ahcyl2 PEST domains in NBCe1-B activation.(A~I) HeLa cells were transfected with GFP-NBCe1-B and an empty vector or vectors expressing Ahcyl2 or its point or PEST domain deletion mutant, (J) NBCe1-B activity was measured and normalized relative to control. Bars indicate mean±SEM of 7~10 separate experiments. *p<0.01 versus NBCe1-B. Numbers below the labels on the x-axis indicate the frequen cy of experiments.


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