Structural characterization of a neuroblast-specific phosphorylated region of MARCKS

Luzineide W. Tinoco, Jully L. Fraga, Cristiane D. AnoBom, Flavio R. Zolessi, Gonzalo Obal, Andrea Toledo, Otto Pritsch, Cristina Arruti
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 2014-04-01; 1844(4): 837-849
DOI: 10.1016/j.bbapap.2014.02.016

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1. Biochim Biophys Acta. 2014 Apr;1844(4):837-49. doi: 10.1016/j.bbapap.2014.02.016.
Epub 2014 Feb 28.

Structural characterization of a neuroblast-specific phosphorylated region of
MARCKS.

Tinoco LW(1), Fraga JL(2), Anobom CD(3), Zolessi FR(4), Obal G(5), Toledo A(6),
Pritsch O(7), Arruti C(8).

Author information:
(1)Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de
Janeiro, Cidade Universitária, CCS, Bloco H, Rio de Janeiro 21941-902, RJ,
Brazil. Electronic address: .
(2)Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de
Janeiro, Cidade Universitária, CCS, Bloco H, Rio de Janeiro 21941-902, RJ,
Brazil. Electronic address: .
(3)Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio
de Janeiro, Cidade Universitária, CT, Bloco A, Rio de Janeiro 21941-909, RJ,
Brazil. Electronic address: .
(4)Laboratorio de Cultivo de Tejidos, Sección Biología Celular, DBCM, Facultad de
Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay.
Electronic address: .
(5)Unidad de Biofísica de Proteínas, Institut Pasteur de Montevideo, Mataojo
2020, 11400 Montevideo, Uruguay. Electronic address: .
(6)Laboratorio de Cultivo de Tejidos, Sección Biología Celular, DBCM, Facultad de
Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay.
Electronic address: .
(7)Unidad de Biofísica de Proteínas, Institut Pasteur de Montevideo, Mataojo
2020, 11400 Montevideo, Uruguay. Electronic address: .
(8)Laboratorio de Cultivo de Tejidos, Sección Biología Celular, DBCM, Facultad de
Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay.
Electronic address: .

MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded
protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane
lipids. Such interactions occur at a highly conserved region that is specifically
phosphorylated by PKC: the Effector Domain. There are two other conserved
domains, MH1 (including a myristoylation site) and MH2, also located in the amino
terminal region and whose structure and putative protein binding capabilities are
currently unknown. MH2 sequence contains a serine that we described as being
phosphorylated only in differentiating neurons (S25 in chick). Here, Circular
Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to
characterize the phosphorylated and unphosphorylated forms of a peptide with the
MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is
recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that
S25 phosphorylation does not cause extensive modifications in the peptide
structure. However, the sharper lines, the absence of multiple spin systems and
relaxation dispersion data observed for the phosphorylated peptide suggested a
more ordered structure. Surface Plasmon Resonance was employed to compare the
binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb
3C3 binds to the whole protein and the peptide with a similar affinity, albeit
different kinetics. The slightly ordered structure of the phosphorylated peptide
might be at the origin of its ability to interact with mAb 3C3 antibody, but this
binding did not noticeably modify the peptide structure.

Copyright © 2014 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.bbapap.2014.02.016
PMID: 24590112 [Indexed for MEDLINE]


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