Abstract
Pyridoxal 5'-phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ-aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca2+ - and integrin-binding protein 1 (CIB1) as a PDXP interactor by yeast two-hybrid screening and find a calmodulin (CaM)-binding motif that overlaps with the PDXP-CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca2+ concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP-CIB1 complex may functionally differ from the PDXP-Ca2+ -CaM complex.
Highlights
Pyridoxal 50-phosphate (PLP) is an essential cofactor for neurotransmitter metabolism
Elevated Ca2+ concentrations promote CaM binding and, thereby, diminish Ca2+- and integrin-binding protein 1 (CIB1) binding to Pyridoxal phosphatase (PDXP), as both interactors bind in a mutually exclusive way
Increased Ca2+ levels promote CaM binding to PDXP, and competitively diminish CIB1 binding to PDXP, the CIB1-PDXP association per se is not dependent on changes in [Ca2+]
Summary
Murine PDXP was reverse-transcribed from adult mouse brain cDNA. Total RNA was isolated using TRIzol (Invitrogen/Life Technologies GmbH, Darmstadt, Germany) according to the manufacturer’s instructions, and cDNA was obtained with the High Fidelity RNA PCR Kit (Takara Bio Europe SAS, Saint-Germain-en-Laye, France) and oligo dT primers. Cells were lysed as described above in interaction buffer (without DTT); lysates were incubated with a-CIB1 antibodies for 1 h at 4 °C, and protein A beads (GE Healthcare) were used for immunoprecipitation. Beads were washed three times in wash buffer (20 mM HEPES, 500 mM NaCl, 1% Triton X-100; pH 7.4), and associated proteins were solubilized in Laemmli’s buffer, separated by SDS/PAGE, and analyzed by probing with CIB1- or PDXP-specific antibodies. Five micrometers of PDXP was incubated for 2 h at 37 °C with 10 μM of CIB1 and/or 10 μM of CaM (all proteins were untagged) in a total volume of 15 μL in buffer A supplemented with 50 μM EGTA Æ different concentrations of CaCl2 and 0.67% formaldehyde. Significant differences in the effect of Ca2+ on CIB1-PDXP or CaM-PDXP-complex formation were analyzed with the Mann–Whitney two-tailed test, using GRAPHPAD PRISM version 4.01
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