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N-myristoylated proteins, key components in intracellular signal transduction systems enabling rapid and flexible cell responses.

Hayashi N, Titani K - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2010)

Bottom Line: Thus, it has been shown that myristoylated proteins in cells regulate the signal transduction between membranes and cytoplasmic fractions.Interestingly, a large portion of the myristoylated proteins thought to take part in signal transduction between membranes and cytoplasmic fractions are included in the predicted myristoylated proteins.On the basis of our recent results, this review will highlight the multifunctional aspects of protein N-myristoylation in brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama-shi, Kanagawa Pref., 226-8501, Japan. nhayashi@bio.titech.ac.jp

ABSTRACT
N-myristoylation, one of the co- or post-translational modifications of proteins, has so far been regarded as necessary for anchoring of proteins to membranes. Recently, we have revealed that N(alpha)-myristoylation of several brain proteins unambiguously regulates certain protein-protein interactions that may affect signaling pathways in brain. Comparison of the amino acid sequences of myristoylated proteins including those in other organs suggests that this regulation is involved in signaling pathways not only in brain but also in other organs. Thus, it has been shown that myristoylated proteins in cells regulate the signal transduction between membranes and cytoplasmic fractions. An algorithm we have developed to identify myristoylated proteins in cells predicts the presence of hundreds of myristoylated proteins. Interestingly, a large portion of the myristoylated proteins thought to take part in signal transduction between membranes and cytoplasmic fractions are included in the predicted myristoylated proteins. If the proteins functionally regulated by myristoylation, a posttranslational protein modification, were understood as cross-talk points within the intracellular signal transduction system, known signaling pathways could thus be linked to each other, and a novel map of this intracellular network could be constructed. On the basis of our recent results, this review will highlight the multifunctional aspects of protein N-myristoylation in brain.

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Related in: MedlinePlus

Effects of myristoylation of recombinant CAP-23/NAP-22 on the interaction with CaM. Binding of the non-myristoylated (A) and myristoylated (B) recombinant CAP-23/NAP-22 to CaM was determined using CaM-agarose. The proteins (Lane 1) were mixed with calmodulin-agarose in 50 mM Tris-HCl buffer (pH 6.8) containing 1 mM CaCl2 and 0.2 M NaCl. After a short period of centrifugation in a tabletop centrifuge, the supernatants were removed (Lane 2). The calmodulin-agarose was washed twice with the same buffer. To the sedimented gels, 50 mM Tris-HCl buffer (pH 6.8) containing 0.2 M NaCl and 5 mM EGTA was added. After centrifugation, the supernatants were removed (Lane 3), and the remaining proteins were eluted with the sample buffer containing 1% SDS (Lane 4). The fractions obtained were analyzed by SDS gel electrophoresis.
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fig03: Effects of myristoylation of recombinant CAP-23/NAP-22 on the interaction with CaM. Binding of the non-myristoylated (A) and myristoylated (B) recombinant CAP-23/NAP-22 to CaM was determined using CaM-agarose. The proteins (Lane 1) were mixed with calmodulin-agarose in 50 mM Tris-HCl buffer (pH 6.8) containing 1 mM CaCl2 and 0.2 M NaCl. After a short period of centrifugation in a tabletop centrifuge, the supernatants were removed (Lane 2). The calmodulin-agarose was washed twice with the same buffer. To the sedimented gels, 50 mM Tris-HCl buffer (pH 6.8) containing 0.2 M NaCl and 5 mM EGTA was added. After centrifugation, the supernatants were removed (Lane 3), and the remaining proteins were eluted with the sample buffer containing 1% SDS (Lane 4). The fractions obtained were analyzed by SDS gel electrophoresis.

Mentions: Interaction of the two recombinant proteins with CaM was analyzed by the binding to CaM-agarose beads (Fig. 3). Clearly, only the myristoylated protein bound to the CaM-beads, and most of the bound protein was eluted with a Ca2+-free buffer. The non-myristoylated protein did not bind to the CaM beads to any significant extent. Therefore, CAP-23/NAP-22 bound to CaM in a Ca2+ and myristoylation-dependent manner. It was also shown that the binding of mC/N9, N-myristoylated 9 residue peptide corresponding to the N-terminal CaM binding site of CAP-23/NAP-22, to CaM was dependent on the existence of the myristoyl moiety (Fig. 4). Furthermore, phosphorylation of Ser5 in the N-terminal region of CAP-23/NAP-22 by PKC abolished the binding of CAP-23/NAP-22 to Ca2+/CaM (Fig. 4).14) This was assumed to be caused by the introduction of an oppositely charged group into the middle of the basic residues that were essential in making the ionic contact with negatively charged CaM.


N-myristoylated proteins, key components in intracellular signal transduction systems enabling rapid and flexible cell responses.

Hayashi N, Titani K - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2010)

Effects of myristoylation of recombinant CAP-23/NAP-22 on the interaction with CaM. Binding of the non-myristoylated (A) and myristoylated (B) recombinant CAP-23/NAP-22 to CaM was determined using CaM-agarose. The proteins (Lane 1) were mixed with calmodulin-agarose in 50 mM Tris-HCl buffer (pH 6.8) containing 1 mM CaCl2 and 0.2 M NaCl. After a short period of centrifugation in a tabletop centrifuge, the supernatants were removed (Lane 2). The calmodulin-agarose was washed twice with the same buffer. To the sedimented gels, 50 mM Tris-HCl buffer (pH 6.8) containing 0.2 M NaCl and 5 mM EGTA was added. After centrifugation, the supernatants were removed (Lane 3), and the remaining proteins were eluted with the sample buffer containing 1% SDS (Lane 4). The fractions obtained were analyzed by SDS gel electrophoresis.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108300&req=5

fig03: Effects of myristoylation of recombinant CAP-23/NAP-22 on the interaction with CaM. Binding of the non-myristoylated (A) and myristoylated (B) recombinant CAP-23/NAP-22 to CaM was determined using CaM-agarose. The proteins (Lane 1) were mixed with calmodulin-agarose in 50 mM Tris-HCl buffer (pH 6.8) containing 1 mM CaCl2 and 0.2 M NaCl. After a short period of centrifugation in a tabletop centrifuge, the supernatants were removed (Lane 2). The calmodulin-agarose was washed twice with the same buffer. To the sedimented gels, 50 mM Tris-HCl buffer (pH 6.8) containing 0.2 M NaCl and 5 mM EGTA was added. After centrifugation, the supernatants were removed (Lane 3), and the remaining proteins were eluted with the sample buffer containing 1% SDS (Lane 4). The fractions obtained were analyzed by SDS gel electrophoresis.
Mentions: Interaction of the two recombinant proteins with CaM was analyzed by the binding to CaM-agarose beads (Fig. 3). Clearly, only the myristoylated protein bound to the CaM-beads, and most of the bound protein was eluted with a Ca2+-free buffer. The non-myristoylated protein did not bind to the CaM beads to any significant extent. Therefore, CAP-23/NAP-22 bound to CaM in a Ca2+ and myristoylation-dependent manner. It was also shown that the binding of mC/N9, N-myristoylated 9 residue peptide corresponding to the N-terminal CaM binding site of CAP-23/NAP-22, to CaM was dependent on the existence of the myristoyl moiety (Fig. 4). Furthermore, phosphorylation of Ser5 in the N-terminal region of CAP-23/NAP-22 by PKC abolished the binding of CAP-23/NAP-22 to Ca2+/CaM (Fig. 4).14) This was assumed to be caused by the introduction of an oppositely charged group into the middle of the basic residues that were essential in making the ionic contact with negatively charged CaM.

Bottom Line: Thus, it has been shown that myristoylated proteins in cells regulate the signal transduction between membranes and cytoplasmic fractions.Interestingly, a large portion of the myristoylated proteins thought to take part in signal transduction between membranes and cytoplasmic fractions are included in the predicted myristoylated proteins.On the basis of our recent results, this review will highlight the multifunctional aspects of protein N-myristoylation in brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama-shi, Kanagawa Pref., 226-8501, Japan. nhayashi@bio.titech.ac.jp

ABSTRACT
N-myristoylation, one of the co- or post-translational modifications of proteins, has so far been regarded as necessary for anchoring of proteins to membranes. Recently, we have revealed that N(alpha)-myristoylation of several brain proteins unambiguously regulates certain protein-protein interactions that may affect signaling pathways in brain. Comparison of the amino acid sequences of myristoylated proteins including those in other organs suggests that this regulation is involved in signaling pathways not only in brain but also in other organs. Thus, it has been shown that myristoylated proteins in cells regulate the signal transduction between membranes and cytoplasmic fractions. An algorithm we have developed to identify myristoylated proteins in cells predicts the presence of hundreds of myristoylated proteins. Interestingly, a large portion of the myristoylated proteins thought to take part in signal transduction between membranes and cytoplasmic fractions are included in the predicted myristoylated proteins. If the proteins functionally regulated by myristoylation, a posttranslational protein modification, were understood as cross-talk points within the intracellular signal transduction system, known signaling pathways could thus be linked to each other, and a novel map of this intracellular network could be constructed. On the basis of our recent results, this review will highlight the multifunctional aspects of protein N-myristoylation in brain.

Show MeSH
Related in: MedlinePlus