PROTEIN PHOSPHORYLATION IN NEURONAL FUNCTION 
Thomas R. Soderung, Ph.D., Investigator 
The major focus of the research in Dr. Soderling's 
laboratory this past year has been further elucida- 
tion of the regulatory mechanisms of Ca^"*"/ 
calmodulin-dependent protein kinase II (CaM-ki- 
nase II). This multifunctional protein kinase is 
highly abundant in brain, especially hippocampus, 
where it constitutes —1-2% of total protein and 
30-50% of the postsynaptic density protein. This 
kinase is thought to be involved in numerous neu- 
ronal functions, including regulation of neurotrans- 
mitter synthesis and exocytosis and perhaps in syn- 
aptic plasticity such as long-term potentiation. 
The regulatory consequences of autophosphor- 
ylation of Thr-286 in the dodecameric holoenzyme 
were described last year: namely, conversion of 
the kinase to a partially Ca^"'"-independent species. 
The essential role of Thr-286 has now been con- 
firmed by mutation to either Ala or Asp. The Ala- 
286 mutant was just like the wild-type kinase, ex- 
cept that autophosphorylation did not generate a 
Ca^^-independent form. The Asp-286 mutant 
kinase already exhibited Ca^^-independent activity 
prior to autophosphorylation. This indicates that 
the presence of the negative charge at residue 286, 
either by phosphorylation of the Thr or by mu- 
tation to Asp, is sufficient for Ca^^-independent 
activity. 
The autoinhibitory domain in CaM-kinase II (res- 
idues 281-309) contains multiple regulatory ele- 
ments. Residues 296-309 constitute the CaM-bind- 
ing domain, residues 290-302 are inhibitory 
through interaction with the substrate protein- 
binding site, and residues 281-290 potentiate the 
inhibition by interfering with the ATP-binding site. 
PUBLICATIONS 
The synthetic peptide containing residues 281-309 
is a strong inhibitor of CaM-kinase II {K^ = 0.2 |jlM). 
The inhibitory potency of the synthetic peptide is 
completely reversed by binding of Ca^^/CaM. Phos- 
phorylation of Thr-286 greatly decreases its inhibi- 
tory potency but does not affect the binding of 
Ca^^/CaM. On the other hand, phosphorylation of 
Thr-305 or Thr-306 prevents binding of Ca^VCaM. 
These studies support the following model for 
the regulation of brain CaM-kinase II. In the inac- 
tive form, the autoinhibitory domain interacts with 
the catalytic binding sites for both protein and ATE 
Binding of Ca^^/CaM to residues 296-309 induces 
a conformational change in the overlapping inhibi- 
tory region (residues 281-302), thereby disrupting 
the interaction of the autoinhibitory domain with 
the catalytic site and activating the kinase. The acti- 
vated kinase can phosphorylate exogenous protein 
substrates that contain the consensus recognition 
sequence (-R-R-X-S/T) as well as autophosphoryla- 
tion on Thr-286. The autophosphorylated kinase is 
active in the absence of Ca^"'"/CaM, since the nega- 
tive charge at residue 286 prevents the autoinhibi- 
tory domain from interacting with the catalytic site. 
The Ca^"'^-independent form undergoes further au- 
tophosphorylation on Thr-305 -306 within the 
CaM-binding domain, which prevents binding of 
Ca^''^/CaM. This complex regulatory mechanism for 
CaM-kinase II presumably allows it to perform 
multiple modulatory roles in neuronal functions. 
Dr. Soderling is Professor of Molecular Physiology 
and Biophysics at the Vanderbilt University School 
of Medicine. 
Articles 
Colbran, R.J., Fong, Y-L., Schworer, CM., and Soderling, TR. 1988. Regulatory interactions between the 
calmodulin-binding, inhibitory, and autophosphorylation domains of Ca^"*" /calmodulin-dependent protein 
kinase II. J Biol Chem 263:18145-18151. 
Colbran, R.J. , Schworer, CM. , Hashimoto, Y , Fong, Y-L., Rich, D.P , Smith, A.K., and Soderling. TR. 1989. Cal- 
cium/calmodulin-dependent protein kinase II. Biochem /258:313-325. 
Colbran, R.J. , Smith, K.M., Schworer, CM. , Fong, Y-L., and Soderling, TR. 1989. Regulatory domain of cal- 
cium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphoryla- 
tion. J Biol Chem 264:4800-4804. 
Hashimoto, Y , King, M.M., and Soderling, TR. 1988. Regulatory interactions of calmodulin-binding proteins: 
phosphorylation of calcineurin by autophosphorylated Ca^ "'"/calmodulin-dependent protein kinase II. 
Proc Natl Acad Sci USA 85:7001-7005. 
Continued 
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