Schleiff E, Schmitz A, McIlhinney R A, Manenti S, Vergères G
Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.
J Biol Chem. 1996 Oct 25;271(43):26794-802. doi: 10.1074/jbc.271.43.26794.
The members of the myristoylated alanine-rich C kinase substrate (MARCKS) family are proteins essential for brain development and phagocytosis. MARCKS proteins bind to actin filaments and calmodulin (CaM) and are phosphorylated by protein kinase C. In order to investigate how these interactions are regulated, we have characterized the properties of both the myristoylated (myr) and unmyristoylated (unmyr) forms of recombinant MARCKS-related protein (MRP), a 20-kDa member of the MARCKS family. Ultracentrifugation and circular dichroic spectroscopy reveal that MRP is an elongated protein, with an axis ratio estimated between 7 and 12 and with an apparent random coil conformation. MRP binds to CaM with high affinity (Kd,myr = 4 nM; Kd,unmyr = 7 nM) and with a second order rate constant, k+1,unmyr, of 1.6 x 10(8) M-1 s-1. In contrast to classical ligands such as the myosin light chain kinase, binding of MRP to CaM does not induce the formation of an alpha-helix in MRP. The catalytic subunit of protein kinase C (PKM) phosphorylates myr MRP with high affinity ([S]0.5 = 3.5 microM), positive cooperativity (nH = 2.5) and a turnover number of 130 min-1. CaM inhibits the phosphorylation of myr MRP with a half-maximum rate of phosphorylation at a [CaM]/[MRP] ratio of 0.7, indicating that CaM might efficiently regulate the phosphorylation of MRP in vivo. Interestingly, Ca2+ inhibits the binding of MRP to CaM as well as its phosphorylation by PKM in the millimolar concentration range, suggesting that MRP has a weak affinity for Ca2+. Finally, unmyr MRP can be stoichiometrically myristoylated by N-myristoyl transferase in vitro. Since neither binding of CaM nor phosphorylation by PKM inhibits myristoylation, the N terminus of unmyr MRP is exposed on the surface of the protein and is well separated from the effector domain. In view of the observations that unmyr and myr MRP do not exhibit significant differences in their properties in solution, the function of myristoylation is most probably to modulate the interactions of MRP with membranes.
富含豆蔻酰化丙氨酸的C激酶底物（MARCKS）家族成员是大脑发育和吞噬作用所必需的蛋白质。MARCKS蛋白与肌动蛋白丝和钙调蛋白（CaM）结合，并被蛋白激酶C磷酸化。为了研究这些相互作用是如何被调控的，我们对重组MARCKS相关蛋白（MRP）的豆蔻酰化（myr）和非豆蔻酰化（unmyr）形式的特性进行了表征，MRP是MARCKS家族中一个20 kDa的成员。超速离心和圆二色光谱表明，MRP是一种细长的蛋白质，轴比估计在7到12之间，具有明显的无规卷曲构象。MRP与CaM以高亲和力结合（Kd,myr = 4 nM；Kd,unmyr = 7 nM），二级速率常数k+1,unmyr为1.6 x 10(8) M-1 s-1。与诸如肌球蛋白轻链激酶等经典配体不同，MRP与CaM的结合不会在MRP中诱导α-螺旋的形成。蛋白激酶C（PKM）的催化亚基以高亲和力（[S]0.5 = 3.5 microM）、正协同性（nH = 2.5）和130 min-1的周转数对myr MRP进行磷酸化。CaM以[CaM]/[MRP]比值为0.7时的磷酸化半最大速率抑制myr MRP的磷酸化，表明CaM可能在体内有效地调节MRP的磷酸化。有趣的是，Ca2+在毫摩尔浓度范围内抑制MRP与CaM的结合以及PKM对其的磷酸化，这表明MRP对Ca2+具有弱亲和力。最后，unmyr MRP在体外可被N-豆蔻酰转移酶化学计量地豆蔻酰化。由于CaM的结合和PKM的磷酸化均不抑制豆蔻酰化，unmyr MRP的N末端暴露在蛋白质表面，且与效应结构域相距甚远。鉴于观察到unmyr和myr MRP在溶液中的性质没有显著差异，豆蔻酰化的功能很可能是调节MRP与膜的相互作用。
