Wong E V, Schaefer A W, Landreth G, Lemmon V
Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106-4975, USA.
J Neurochem. 1996 Feb;66(2):779-86. doi: 10.1046/j.1471-4159.1996.66020779.x.
L1 is an axonal cell adhesion molecule found primarily on projection axons of both the CNS and PNS. It is a phosphorylated membrane-spanning glycoprotein that can be immunoprecipitated from rat brain membranes in association with protein kinase activities. Western blot analysis demonstrates that casein kinase II (CKII), a ubiquitous serine/threonine kinase enriched in brain, is present in these immunoprecipitates. CKII preparations partially purified from PC12 cells are able to phosphorylate recombinant L1 cytoplasmic domain (L1CD), which consists of residues 1,144-1,257. Using these as well as more highly purified kinase preparations, phosphorylation assays of small peptides derived from the L1CD were performed. CKII was able to phosphorylate a peptide encompassing amino acids (aa) 1,173-1,185, as well as a related peptide representing an alternatively spliced nonneuronal L1 isoform that lacks aa 1,177-1,180. Both peptides were phosphorylated with similar kinetic profiles. Serine to alanine substitutions in these peptides indicate that the CKII phosphorylation site is at Ser1,181. This is consistent with experiments in which L1CD was phosphorylated by these kinase preparations, digested, and the radiolabeled fragments sequenced. Furthermore, when L1 immunoprecipitates were used to phosphorylate L1CD, one of the residues phosphorylated is the same residue phosphorylated by CKII. Finally, in vivo radiolabeling indicates that Ser1,181 is phosphorylated in newborn rat brain. These data show that CKII is associated with and able to phosphorylate L1. This phosphorylation may be important in regulating certain aspects of L1 function, such as adhesivity or signal transduction.
L1是一种轴突细胞粘附分子，主要存在于中枢神经系统和周围神经系统的投射轴突上。它是一种磷酸化的跨膜糖蛋白，可与蛋白激酶活性相关联，从大鼠脑膜中免疫沉淀出来。蛋白质印迹分析表明，酪蛋白激酶II（CKII），一种在大脑中富集的普遍存在的丝氨酸/苏氨酸激酶，存在于这些免疫沉淀物中。从PC12细胞中部分纯化的CKII制剂能够磷酸化重组L1细胞质结构域（L1CD），该结构域由1144 - 1257位残基组成。利用这些以及更高纯度的激酶制剂，对源自L1CD的小肽进行了磷酸化测定。CKII能够磷酸化包含氨基酸（aa）1173 - 1185的肽段，以及代表缺乏aa 1177 - 1180的选择性剪接的非神经元L1同种型的相关肽段。两种肽段的磷酸化具有相似的动力学特征。这些肽段中丝氨酸到丙氨酸的取代表明CKII磷酸化位点在Ser1181。这与L1CD被这些激酶制剂磷酸化、消化并对放射性标记片段进行测序的实验结果一致。此外，当使用L1免疫沉淀物对L1CD进行磷酸化时，磷酸化的残基之一与CKII磷酸化的残基相同。最后，体内放射性标记表明Ser1181在新生大鼠脑中被磷酸化。这些数据表明CKII与L1相关联并能够使其磷酸化。这种磷酸化可能在调节L1功能的某些方面，如粘附性或信号转导中起重要作用。
