The influence of phosphorylation on the activity and structure of the neuronal IQ motif protein, PEP-19.
PEP-19 is a 7.6 kDa neuronally expressed polypeptide that contains a single calmodulin-binding IQ motif. The calmodulin-binding activity of several neuronal IQ motif proteins is regulated by phosphorylation of a conserved serine. We propose that the serine residue within the IQ motif of PEP-19 is phosphorylated, and that phosphorylation modifies the activity of PEP-19. Camstatin, a functionally active 25-residue fragment of PEP-19's IQ motif, binds calmodulin and inhibits neuronal nitric oxide synthase. A truncated camstatin-in which the IQ motif serine is the only phosphorylatable residue-was screened against 42 different kinases. Truncated camstatin is selectively phosphorylated by four isoforms of protein kinase C. Furthermore, treatment of full-length PEP-19 with PKCgamma catalyzes phosphorylation of the same serine residue. Fluorescent anisotropy shows that phosphorylation of camstatin inhibits its binding to calmodulin. NMR solution structures indicate that both camstatin and phospho-camstatin exist in similar dynamic turn-like conformations. This suggests that camstatin's greater affinity for calmodulin is due not to a change in the conformation of the phospho-peptide, but rather, to a disruption of hydrophobic interactions between phospho-camstatin and calmodulin caused by the presence of the hydrophilic phosphate group. The H(alpha) chemical shifts and the circular dichroism spectra of the camstatins are consistent with those of "nascent helices". We submit that PEP-19 is a PKC substrate, and that the phosphorylation state of PEP-19 may play a role in the modulation of calmodulin-dependent signaling.
Dickerson
J Bradley
JB
Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Morgan
Marc A
MA
Mishra
Ashutosh
A
Slaughter
Clive A
CA
Morgan
James I
JI
Zheng
Jie
J
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence / Q000502:physiology; D005454:Fluorescence Polarization; D006801:Humans; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D002147:Calmodulin; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; C513477:camstatin; C050394:PCP4 protein, human; D012694:Serine; D011493:Protein Kinase C