James G, Olson E N
Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
J Biol Chem. 1989 Dec 15;264(35):20928-33.
Numerous reports have described a phosphoprotein with an apparent molecular mass of 68-87 kDa, often referred to as the 80K protein, which serves as a major specific substrate for protein kinase C in a wide variety of cell types. This protein has been shown to be myristoylated in macrophages, apparently in a stimulus-dependent manner. In the present study, we have defined the kinetics for myristoylation of the 80K protein in BC3H1 myocytes and have examined the subcellular distribution of the [3H]myristate and 32P-labeled forms of the protein before and after activation of protein kinase C by phorbol dibutyrate (PDBu). The 80K protein was identified in BC3H1 myocytes by apparent molecular mass of 68 kDa (consistent with the previously reported size of the murine homologue), isoelectric point of 4.6-4.8, PDBu-inducible phosphorylation, peptide mapping, and labeling with [3H]myristate. Incorporation of [3H]myristate by this protein occurred through an amide linkage and was abolished completely by cycloheximide. Pulse labeling of quiescent cells with [3H]myristate revealed no alteration in myristoylation of the 80K protein in either the crude membrane or soluble fractions after PDBu-induced phosphorylation. The subcellular distribution of this protein (approximately 80% membrane, approximately 20% cytosol) also was the same in control and PDBu-stimulated cells. Phosphorylation of both the membrane-bound and soluble forms was increased approximately 6-fold upon stimulation of cultures with PDBu; the soluble form was phosphorylated to a 4-fold higher stoichiometry than its membrane-bound counterpart. Together, these data demonstrate that the 80K protein is myristoylated cotranslationally in BC3H1 cells and that protein kinase C-dependent phosphorylation of the 80K protein does not alter its subcellular distribution or degree of myristoylation. The fact that 20% of total myristoylated 80K protein resides in the cytosol also indicates that myristoylation alone is not sufficient to target this protein to the plasma membrane.
众多报告描述了一种表观分子量为68 - 87 kDa的磷蛋白，常被称为80K蛋白，它在多种细胞类型中作为蛋白激酶C的主要特异性底物。该蛋白已被证明在巨噬细胞中发生肉豆蔻酰化，显然是以刺激依赖的方式。在本研究中，我们确定了BC3H1肌细胞中80K蛋白肉豆蔻酰化的动力学，并研究了在用佛波酯二丁酯（PDBu）激活蛋白激酶C前后，该蛋白的[3H]肉豆蔻酸盐和32P标记形式的亚细胞分布。通过表观分子量68 kDa（与先前报道的小鼠同源物大小一致）、等电点4.6 - 4.8、PDBu诱导的磷酸化、肽图谱分析以及用[3H]肉豆蔻酸盐标记，在BC3H1肌细胞中鉴定出了80K蛋白。该蛋白对[3H]肉豆蔻酸盐的掺入通过酰胺键进行，并且完全被环己酰亚胺抑制。用[3H]肉豆蔻酸盐对静止细胞进行脉冲标记显示，在PDBu诱导的磷酸化后，粗膜或可溶性部分中80K蛋白的肉豆蔻酰化没有改变。该蛋白的亚细胞分布（约80%在膜上，约20%在胞质溶胶中）在对照细胞和PDBu刺激的细胞中也相同。在用PDBu刺激培养物后，膜结合形式和可溶性形式的磷酸化均增加了约6倍；可溶性形式的磷酸化化学计量比其膜结合对应物高4倍。总之，这些数据表明80K蛋白在BC3H1细胞中是共翻译肉豆蔻酰化的，并且80K蛋白的蛋白激酶C依赖性磷酸化不会改变其亚细胞分布或肉豆蔻酰化程度。总肉豆蔻酰化的80K蛋白中有20%存在于胞质溶胶中这一事实也表明，仅肉豆蔻酰化不足以将该蛋白靶向到质膜。
