Abstract
The protein kinase C (PKC)-mediated phosphorylation of the Na+/K+-ATPase α-subunit has been shown to play an important role in regulation of the Na+/K+-ATPase activity. In the rat α1-subunit, phosphorylation occurs at Ser-23 and results in inhibition of the transport function of the Na+/K+-ATPase, which is mimicked by replacing the Ser-23 by the negatively charged glutamic acid or by aspartic acid. Using comparative molecular modeling, we investigated whether phosphorylation or acidic replacement at position 23 causes a dramatic change in the molecular electrostatic potential at position 23 as a result of insertion of a negative charge of the phosphoryl group or Glu per se, or whether, alternatively, the modification causes larger-scale conformational changes in the N-terminus of the α-subunit. The results predict a considerable conformational change of the 30-residue stretch around Ser-23 when mutated to the residues carrying a net negative charge or being phosphorylated. The structural rearrangements occur within the N-terminal helix-loop-helix motif with a set of charged residues. This motif has structural homology with one in the Ca2+-ATPase and may form a function-related structural site in the P-type ATPases. Comparative molecular modeling indicates a lengthening of the interhelical loop and an order-to-disorder transition by disrupting a helix at position 23 because of posphorylation.
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References
Therien, A. G. and Blostein, R. (2000) Mechanisms of sodium pump regulation. Am. J. Physiol Cell Phys. 279, C541-C566.
Bertorello, A. M., Aperia, A., Walaas, S. I., Nairn, A. C., and Greengard, P. (1991) Phosphorylation of the catalytic subunit of Na+, K+-ATPase inhibits the activity of the enzyme. Proc. Natl. Acad. Sci. USA 88, 11,359–11,362.
Vasilets, L. A. (1997) Diversity of regulatory phosphorylation of the Na+/K+-ATPase from mammalian kidneys and Xenopus oocytes by protein kinases. Characterisation of the phosphorylation site for PKC. Cell. Physiol. Biochem. 7, 1–18.
Vasilets, L. A. (2002) Mechanisms of short-term regulation of the Na+/K+-ATPase by protein kinases. Biol. Membr., 19, 77–82.
Feschenko, M. S. and Sweadner, K. J. (1995) Structural basis for species-specific differences in the phosphorylation of Na+, K+-ATPase by protein kinase C. J. Biol. Chem. 270, 14,072–14,077.
Logvinenko, N. S., Dulubova, I., Fedosova, N., Larsson, S. H., Nairn, A. C., Esmann, M., et al. (1996) Phosphorylation by protein kinase C of serine-23 of the α1 subunit of rat Na+, K+-ATPase affects its conformational equilibrium. Proc. Natl. Acad. Sci. USA 93, 9132–9137.
Vasilets, L. A., Spielman, A., and Schwarz, W. (2002) S23E mutation of the PKC phosphorylation site of the α-subunit of Na,K-ATPase leads to increase of surface expression of pumps and reduction of the transport rate. Pflügers Arch. 443, S283.
Vasilets, L. A., Postina, R., and Kirichenko, S. N. (1999) Mutation of Ser-23 of the α1 subunit of the rat Na+/K+-ATPase to negatively charged amino acid residues mimic the functional effect of PKC-mediated phosphorylation. FEBS Lett. 455, 8–12.
Vasilets, L. A., Ohta, T., Noguchi, S., Kawamura, M., and Schwarz, W. (1993) Voltage-dependent inhibition of the sodium pump by external sodium: species differences and possible role of the N-terminus of the α-subunit. Eur. Biophys. J. 21, 433–443.
Vasilets, L. A., Omay, H., Ohta, T., Noguchi, S., Kawamura, M., and Schwarz, W. (1991) Stimulation of the Na+/K+ pump by external [K+] is regulated by voltage-dependent gating. J. Biol. Chem. 266, 16,285–16,288.
Wierzbicki, W. and Blostein, R. (1993) The amino-terminal segment of the catalytic subunit of kidney Na,K-ATPase regulates the potassium deocclusion pathway of the reaction cycle. Proc. Natl. Acad. Sci. USA 90, 70–74.
Daly, S. E., Lane, L. K., and Blostein, R. (1996) Structure/function analysis of the amino-terminal region of the α-1 and α-2 subunits of Na,K-ATPase. J. Biol. Chem. 271, 23,683–23,689.
Vasilets, L. A., Wu, C. H., Wachter, E., and Schwarz, W. (2000) Gating role of the N-terminus of α-subunit of the Na+,K+-ATPase converted into a channel by palytoxin, in Control and Diseases of Sodium Transport Proteins and Channels. (Suketa, Y., ed.), Elsevier, Amsterdam, pp. 23–26.
Vasilets, L. A., Brandt, W., Postina, R., Kirichenko, S., and Anders, A. (2000) Molecular mechanisms of PKC-mediated inhibition of cation transport by the Na+/K+-ATPase: site-directed mutagenesis and molecular modelling studies. Pflügers Arch. 439, R321.
Vasilets, L. A., Brandt, W., Postina, R., Fotis, H., Tatjanenko, L. V., Gvozdev, A. R., et al. (2000) Molecular mechanisms of covalent regulation of the Na+/K+-ATPase by protein kinases, in The Sodium Pump (Taniguchi, K. and Kaya, S., eds.), Elsevier, Amsterdam, pp. 507–572.
http://scop.mrc-lmb.cam.ac.uk/scop/aln.cgi
Altshul, S. F., Madden, T. L., Schäffer, A. A., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.
Zhang, J. and Madden, T. L. (1997) PowerBLAST: a new network BLAST application for interactive or automated sequence analysis and annotation. Genome Res. 7, 649–656.
Pearson, W. R. and Lipman, D. J. (1988) Improved tools for biological sequence analysis. Proc. Natl. Acad. Sci. USA 85, 2444–2448.
Pearson, W. R. (1990) Rapid and sensitive sequence comparison with FASP and FASTA. Methods Enzymol. 183, 63–98.
Tripos, Inc. (1998) SYBYL 6.5 Tripos Inc., St. Louis, Mo.
Clark, M., Cramer, R. D. III, and Van Opendbosch, N. (1989) Validation of the general purpose TRIPOS field. J. Comput. Chem. 10, 982–1012.
Gasteiger, J. and Marsili, M. (1980) Iterative partial equalization of orbital electronegativity, a rapid access to atomic charges. Tetrahedron 36, 3219–3238.
Laskowski, R. A., McArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) PROCHECK: a program to check the stereometrical quality of protein structure. J. Appl. Cryst. 26, 286–289.
Kabsch, W. and Sander, C. (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637.
Thornton, J. M. (2001) From genome to function. Science 292, 2095–2097.
Gaudet, R., Savage, J. R., McLaughlin, J. N., Willardson, B. M., and Sigler, P. B. (1999) A molecular mechanism for the phosphorylation-dependent regulation of heterotrimeric G proteins by phosducin. Mol. Cell 3, 649–660.
Dean, A. M. and Koshland, D. E. (1990) Electrostatic and steric contributions to regulation at the active site of isocitrate dehydrogenase. Science 249, 1044–1046.
Antz, C., Geyer, M., Fakler, B., Schott, M., Guy, H. R., Frank, R., et al. (1997) NMR structure of inactivation gates from mammalian voltage-dependent potassium channels. Nature 385, 272–275.
Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405, 647–655.
Toyoshima, C. and Nomura, H. (2002) Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418, 605–611.
Sweadner, K. J. and Donnet, K. (2001) Structural similarities of Na, K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum. Biochem. J. 356, 685–704.
Hebert, H., Purhonen, P., Vorum, H., Thomsen, K., and Maunsbach, A. B. (2001) Three-dimensional structure of renal Na, K-ATPase from cryo-electron microscopy of two-dimensional crystals. J. Mol. Biol. 314, 479–494.
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Brandt, W., Anders, A. & Vasilets, L.A. Predicted alterations in tertiary structure of the N-terminus of Na+/K+-ATPase α-subunit caused by phosphorylation or acidic replacement of the PKC phosphorylation site Ser-23. Cell Biochem Biophys 37, 83–95 (2002). https://doi.org/10.1385/CBB:37:2:083
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DOI: https://doi.org/10.1385/CBB:37:2:083