Calcium-sensitive interaction between calmodulin and modified forms of rat brain neurogranin/RC3.
Neurogranin (NG) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca(2+) concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the alpha-helical content of this protein. Mutation of the four Cys residues [Cys(-)-NG] to Gly and Ser did not affect the alpha-helical content either. Interaction of CaM with the reduced (red)-, glutathiolated (GS)-, or Cys(-)-NG in the Ca(2+)-free solution resulted in an increase in the alpha-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO(4)-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys(-)-NG > red-NG, GS-NG > ox-NG > PO(4)-NG. Unlike Cys(-)-, red-, and GS-NG, neither ox- nor PO(4)-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism for regulation of its binding affinity to CaM.
Huang
K P
KP
Section on Metabolic Regulation, National Institutes of Health, Bethesda, MD 20892-4510, USA. kphuang@helix.nih.gov
Huang
F L
FL
Li
J
J
Schuck
P
P
McPhie
P
P
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D013058:Mass Spectrometry; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051579:Neurogranin; D009603:Nitroso Compounds / Q000378:metabolism; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D026422:S-Nitrosoglutathione; D014461:Ultracentrifugation
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004220:Disulfides; D009419:Nerve Tissue Proteins; D009603:Nitroso Compounds; C496651:Nrgn protein, rat; D051579:Neurogranin; D026422:S-Nitrosoglutathione; D011493:Protein Kinase C; D005978:Glutathione; D003545:Cysteine; D002118:Calcium