Molloy S S, Kennedy M B
Division of Biology, California Institute of Technology, Pasadena 91125.
Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4756-60. doi: 10.1073/pnas.88.11.4756.
Autophosphorylation of Thr286 on type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) in vitro causes kinase activity to become partially independent of Ca2+. Here we report that Thr286 is the major CaM kinase autophosphorylation site occupied in situ in "organotypic" hippocampal cultures. Measurement of Ca(2+)-independent CaM kinase activity revealed that approximately one-third of the kinase is autophosphorylated in situ when the basal Ca2+ concentration is 15-43 nM. This proportion was substantially reduced 30 min after removal of extracellular Ca2+ or treatment of the cultures with protein kinase inhibitors and was increased by treatment with okadaic acid. Therefore, the high proportion of autophosphorylated kinase at basal Ca2+ concentrations appears to be maintained by Ca(2+)-dependent autophosphorylation. Homogenates of intact hippocampi also contain a high proportion of Ca(2+)-independent type II CaM kinase, 13-23% depending on developmental age. Thus, in hippocampal neurons, an important function of the autophosphorylation mechanism may be to produce a relatively high level of CaM kinase activity, even at basal Ca2+ concentrations, permitting both upward and downward local regulation by physiological agents.
II型钙/钙调蛋白依赖性蛋白激酶（CaM激酶）上的苏氨酸286（Thr286）在体外发生自磷酸化会使激酶活性部分独立于钙离子。在此我们报告，Thr286是“器官型”海马培养物中原位占据的主要CaM激酶自磷酸化位点。对不依赖钙离子的CaM激酶活性的测量显示，当基础钙离子浓度为15 - 43 nM时，约三分之一的激酶在原位发生了自磷酸化。在去除细胞外钙离子或用蛋白激酶抑制剂处理培养物30分钟后，这一比例大幅降低，而用冈田酸处理则使其增加。因此，基础钙离子浓度下高比例的自磷酸化激酶似乎是通过依赖钙离子的自磷酸化来维持的。完整海马体的匀浆中也含有高比例的不依赖钙离子的II型CaM激酶，比例在13% - 23%之间，具体取决于发育年龄。因此，在海马神经元中，自磷酸化机制的一个重要功能可能是即使在基础钙离子浓度下也产生相对高水平的CaM激酶活性，从而允许生理因子进行上调和下调的局部调节。
