Yokoyama C T, Sheng Z H, Catterall W A
Graduate Program in Neurobiology and Behavior, University of Washington, Seattle, Washington 98195, USA.
J Neurosci. 1997 Sep 15;17(18):6929-38. doi: 10.1523/JNEUROSCI.17-18-06929.1997.
The synaptic protein interaction (synprint) site on the N-type calcium channel alpha1B subunit binds to the soluble N-ethylmaleimide-sensitive attachment factor receptor (SNARE) proteins syntaxin and synaptosomal protein of 25 kDa (SNAP-25), and this association may be required for efficient fast synaptic transmission. Protein kinase C (PKC) and calcium and calmodulin-dependent protein kinase type II (CaM KII) phosphorylated a recombinant his-tagged synprint site polypeptide rapidly to a stoichiometry of 3-4 mol of phosphate/mol, whereas cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) phosphorylated the synprint peptide more slowly to a stoichiometry of <1 mol/mol. Two-dimensional phosphopeptide mapping revealed similar patterns of phosphorylation of synprint polypeptides and native rat brain N-type calcium channel alpha1B subunits by PKC and Cam KII. Phosphorylation of the synprint peptide with PKC or CaM KII, but not PKA or PKG, strongly inhibited binding of recombinant syntaxin or SNAP-25, even at a level of free calcium (15 microM) that stimulates maximal binding. In contrast, phosphorylation of syntaxin and SNAP-25 with PKC and CaM KII did not affect interactions with the synprint site. Binding assays with polypeptides representing the N- and C-terminal halves of the synprint site indicate that the PKC- and CaM KII-mediated inhibition of binding involves multiple, disperse phosphorylation sites. PKC or CaM KII phosphorylation of the synprint peptide also inhibited its interactions with native rat brain SNARE complexes containing syntaxin and SNAP-25. These results suggest that phosphorylation of the synprint site by PKC or CaM KII may serve as a biochemical switch for interactions between N-type calcium channels and SNARE protein complexes.
N型钙通道α1B亚基上的突触蛋白相互作用（synprint）位点与可溶性N - 乙基马来酰亚胺敏感附着因子受体（SNARE）蛋白 syntaxin和25 kDa的突触体相关蛋白（SNAP - 25）结合，这种结合可能是高效快速突触传递所必需的。蛋白激酶C（PKC）以及钙调蛋白依赖性蛋白激酶II型（CaM KII）能迅速将重组的带有组氨酸标签的synprint位点多肽磷酸化，磷酸化化学计量比达到3 - 4摩尔磷酸盐/摩尔，而环磷酸腺苷依赖性蛋白激酶（PKA）和环磷酸鸟苷依赖性蛋白激酶（PKG）对synprint肽的磷酸化则较为缓慢，磷酸化化学计量比小于1摩尔/摩尔。二维磷酸肽图谱显示，PKC和CaM KII对synprint多肽和天然大鼠脑N型钙通道α1B亚基的磷酸化模式相似。用PKC或CaM KII而非PKA或PKG对synprint肽进行磷酸化，即使在能刺激最大结合的游离钙水平（15微摩尔）下，也会强烈抑制重组syntaxin或SNAP - 25的结合。相反，用PKC和CaM KII对syntaxin和SNAP - 25进行磷酸化并不影响它们与synprint位点的相互作用。用代表synprint位点N端和C端一半的多肽进行结合试验表明，PKC和CaM KII介导的结合抑制涉及多个分散的磷酸化位点。PKC或CaM KII对synprint肽的磷酸化也抑制了它与含有syntaxin和SNAP - 25的天然大鼠脑SNARE复合物的相互作用。这些结果表明，PKC或CaM KII对synprint位点的磷酸化可能作为N型钙通道与SNARE蛋白复合物之间相互作用的生化开关。
