Protein Ser/Thr phosphatases PPEF interact with calmodulin.
Regulation of protein dephosphorylation by cytoplasmic Ca(2+) levels and calmodulin (CaM) is well established and considered to be mediated solely by calcineurin. Yet, recent identification of protein phosphatases with EF-hand domains (PPEF/rdgC) point to the existence of another group of Ca(2+)-dependent protein phosphatases. We have recently hypothesised that PPEF/rdgC phosphatases might possess CaM-binding sites of the IQ-type in their N-terminal domains. We now employed yeast two-hybrid system and surface plasmon resonance (SPR) to test this hypothesis. We found that entire human PPEF2 interacts with CaM in the in vivo tests and that its N-terminal domain binds to CaM in a Ca(2+)-dependent manner with nanomolar affinity in vitro. The fragments corresponding to the second exons of PPEF1 and PPEF2, containing the IQ motifs, are sufficient for specific Ca(2+)-dependent interaction with CaM both in vivo and in vitro. These findings demonstrate the existence of mammalian CaM-binding protein Ser/Thr phosphatases distinct from calcineurin and suggest that the activity of PPEF phosphatases may be controlled by Ca(2+) in a dual way: via C-terminal Ca(2+)-binding domain and via interaction of the N-terminal domain with CaM.
Kutuzov
Mikhail A
MA
Laboratoire de Biophysique Moléculaire et Cellulaire URA CNRS No. 520, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 38054 Grenoble cedex 9, France. m.kutuzov@usa.net
Solov'eva
Olga V
OV
Andreeva
Alexandra V
AV
Bennett
Nelly
N
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D017124:Conserved Sequence; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
D002147:Calmodulin; C107424:PPEF1 protein, human; D010749:Phosphoprotein Phosphatases; D002118:Calcium