Protein kinase C-mediated phosphorylation and calmodulin binding of recombinant myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein

The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein kinase C (PKC) substrates that also bind calmodulin in a manner regulated by phosphorylation by PKC. The kinetics of PKC-mediated phosphorylation and the calmod...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 1994-03, Vol.269 (12), p.9361-9367
Main Authors: Verghese, G M, Johnson, J D, Vasulka, C, Haupt, D M, Stumpo, D J, Blackshear, P J
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Calmodulin - metabolism
Calmodulin-Binding Proteins
Humans
Intracellular Signaling Peptides and Proteins
Membrane Proteins
Mice
Microfilament Proteins
Molecular Sequence Data
Myristoylated Alanine-Rich C Kinase Substrate
Peptide Mapping
Peptides - chemistry
Phosphorylation
Protein Kinase C - metabolism
Proteins - metabolism
Recombinant Proteins
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein kinase C (PKC) substrates that also bind calmodulin in a manner regulated by phosphorylation by PKC. The kinetics of PKC-mediated phosphorylation and the calmodulin binding properties of intact, recombinant MARCKS and MRP were investigated and compared with previous studies of synthetic peptides spanning the PKC phosphorylation site/calmodulin binding domains (PSCBD) of these proteins. Both MARCKS and MRP were high affinity substrates for the catalytic fragment of PKC, and their phosphorylation occurred with positive cooperativity (MARCKS: S0.5 = 100 nM, KH = 1.43; MRP: S0.5 = 238 nM, KH = 1.72). These affinities are similar to the values determined from studies of their respective PSCBD peptides. Two-dimensional mapping of MRP and its synthetic PSCBD peptide yielded identical patterns of tryptic phosphopeptides, indicating that, as in the case of MARCKS, all of the PKC phosphorylation sites in MRP lie within the 24-amino acid PSCBD. Sequence analysis of tryptic phosphopeptides revealed that the first and third, but not the second, serines in the MRP PSCBD were phosphorylated by PKC. Both MARCKS and MRP bound dansyl-calmodulin with high affinity, with a Kapp of 4.6 and 9.5 nM, respectively. Phosphorylation of MARCKS and MRP by PKC disrupted the protein-calmodulin complexes, with half-lives of 4.0 and 3.5 min, respectively. These studies suggest that intact, recombinant MARCKS and MRP are accurately modeled by their synthetic PSCBD peptides with respect to PKC phosphorylation kinetics and their phosphorylation-dependent calmodulin binding properties.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)37116-8