Phosphorylation of the IQ Domain Regulates the Interaction between Ca2+-vector Protein and Its Target in Amphioxus

Tatiana V Petrova,Takashi Takagi,Jos A Cox

doi:10.1074/jbc.271.43.26646

Tatiana V Petrova, Takashi Takagi + Show 1 more

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https://doi.org/10.1074/jbc.271.43.26646

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Abstract

Calcium vector protein target (CaVPT), a 26-kDa endogenous target of calcium vector protein from Amphioxus (CaVP), contains three distinct regions: a N-terminal Pro-Ala-Lys-rich motif, segment 36-50 displaying sequence similarity to the calmodulin-binding site in neuromodulin and neurogranin where they are designated as the IQ domain; and two immunoglobulin-like folds. The phosphorylation by protein kinase C of Ser-43 in the IQ domain drastically decreases the affinity of CaVPT for CaVP and CaVP protects CaVPT from phosphorylation. Phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase has a similar effect, but in addition to Ser-43 four other phosphorylated sites were identified. Removal of the Pro-Ala-Lys-rich region and the IQ domain in CaVPT by trypsin leads to the loss of binding to CaVP, whereas the chymotryptic fragment, containing these regions and first immunoglobulin-like domain, retained the ability to interact with CaVP. A synthetic IQ domain alone interacts strongly with calmodulin, but not with CaVP. Two main conclusions can be drawn from this study: 1) the regulation of interaction between CaVP and CaVPT is very similar to the mechanism observed in the complex between neuromodulin or neurogranin and calmodulin; 2) in spite of this similarity the entire CaVP-binding site is not restricted to the IQ domain; in addition the Pro-Ala-Lys-rich motif may be necessary for high affinity binding to CaVP.

Highlights

Calcium vector protein (CaVP)1 and its endogenous target (CaVPT) are present in muscle of Amphioxus at unusually high concentration, namely 50 to 100 ␮M in terms of intracellular water, comparable to that of the abundant muscle component troponin C (Cox, 1986; Head and Perry, 1974)
1 The abbreviations used are: CaVP, calcium vector protein; CaVPT, calcium vector protein target; CaM, calmodulin; IQ peptide, synthetic peptide corresponding to the residues 33– 61 of CaVPT; DTT, dithiothreitol; HPLC, high pressure liquid chromatography; PKA, cyclic AMPdependent protein kinase; PKC, protein kinase C; Ig-fold, immunoglobulin-like fold; C1–186, chymotryptic fragment corresponding to residues 1–186 of CaVPT; T51–243, tryptic fragment corresponding to residues 51–243 of CaVPT; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)-1-propanesulfonate; MES, 4-morpholineethanesulfonic acid
The only known endogenous target of CaVP is CaVPT, a unique 26-kDa protein displaying the following domain structure: 1) segment 1–23, a Pro-Ala-Lys-rich repetitive motif at the N terminus; 2) segment 36 –50 displaying sequence similarity to CaM-binding sites in neuromodulin and neurogranin (Alexander et al, 1988; Apel et al, 1990; Baudier et al, 1991) where they are designated as the IQ domains; 3) segment 63–243 composed of two Ig-folds, structural motifs found in myosin light chain kinase (Olson et al, 1990), telokin (Ito et al, 1989), C-protein (Furst et al, 1992), twitchin (Benian et al, 1989), and titin (Labeit et al, 1990), proteins believed to be involved in homotypic interactions as well as in the interaction with myosin

Summary

The abbreviations used are

CaVP, calcium vector protein; CaVPT, calcium vector protein target; CaM, calmodulin; IQ peptide, synthetic peptide corresponding to the residues 33– 61 of CaVPT; DTT, dithiothreitol; HPLC, high pressure liquid chromatography; PKA, cyclic AMPdependent protein kinase; PKC, protein kinase C; Ig-fold, immunoglobulin-like fold; C1–186, chymotryptic fragment corresponding to residues 1–186 of CaVPT; T51–243, tryptic fragment corresponding to residues 51–243 of CaVPT; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)-1-propanesulfonate; MES, 4-morpholineethanesulfonic acid. Our previous study indicated that while the complex CaVPCaVPT possesses a rather rigid structure and does not display pronounced interactive properties, CaVP and CaVPT have characteristics of highly dynamic proteins (Petrova et al, 1995a). This allowed us to hypothesize that the complex represents a non-interactive end product and that CaVPT itself is the relay protein toward the unknown cellular response element. In search of a physiological mechanism of CaVP-CaVPT dissociation we have studied the effect of phosphorylation by PKA and PKC on the interactive properties of CaVPT, in analogy with the dissociation mechanism proposed for neuromodulin/neurogranin and CaM (Apel et al, 1990; Baudier et al, 1991). We studied the interaction of CaVP with a synthetic peptide IQ comprising the IQ domain of CaVPT, in order to evaluate whether this sequence represents the CaVPbinding site, as it was suggested earlier (Takagi and Cox, 1991)

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