Maizels E T, Peters C A, Kline M, Cutler R E, Shanmugam M, Hunzicker-Dunn M
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA.
Biochem J. 1998 Jun 15;332 ( Pt 3)(Pt 3):703-12. doi: 10.1042/bj3320703.
Small heat-shock proteins (sHSPs) are widely expressed 25-28 kDa proteins whose functions are dynamically regulated by phosphorylation. While recent efforts have clearly delineated a stress-responsive p38 mitogen-activated protein-kinase (MAPK)-dependent kinase pathway culminating in activation of the heat-shock (HSP)-kinases, mitogen-activated protein-kinase-activated protein kinase-2 and -3, not all sHSP phosphorylation events can be explained by the p38 MAPK-dependent pathway. The contribution of protein kinase C (PKC) to sHSP phosphorylation was suggested by early studies but later questioned on the basis of the reported poor ability of purified PKC to phosphorylate sHSP in vitro. The current study re-evaluates the role of PKC in sHSP phosphorylation in the light of the isoform complexity of the PKC family. We evaluated the sHSP phosphorylation status in rat corpora lutea obtained from two stages of pregnancy, mid-pregnancy and late-pregnancy, which express different levels of the novel PKC isoform, PKC-delta. Two-dimensional Western blot analysis showed that HSP-27 was more highly phosphorylated in vivo in corpora lutea of late pregnancy, corresponding to the developmental stage in which PKC-delta is abundant and active. Late-pregnant luteal extracts contained a lipid-sensitive HSP-kinase activity which exactly co-purified with PKC-delta using hydroxyapatite and S-Sepharose column chromatography. To determine whether there might be preferential phosphorylation of sHSP by a particular PKC isoform, purified recombinant PKC isoforms corresponding to those PKC isoforms detected in rat corpora lutea were evaluated for HSP-kinase activity in vitro. Recombinant PKC-delta effectively catalysed the phosphorylation of sHSP in vitro, and PKC-alpha was 30-50% as effective as an HSP-kinase; other PKCs tested (beta1, beta2, epsilon and zeta) were poor HSP-kinases. These results show that select PKC family members can function as direct HSP-kinases in vitro. Moreover, the observation of enhanced luteal HSP-27 phosphorylation in vivo, in late pregnancy, when PKC-delta is abundant and active, suggests that select PKC family members contribute to sHSP phosphorylation events in vivo.
小分子热休克蛋白（sHSPs）是广泛表达的25 - 28 kDa蛋白质，其功能受磷酸化动态调控。虽然最近的研究已明确描绘出一条应激反应性的p38丝裂原活化蛋白激酶（MAPK）依赖性激酶途径，最终导致热休克（HSP）激酶、丝裂原活化蛋白激酶激活的蛋白激酶-2和-3的激活，但并非所有sHSP磷酸化事件都能通过p38 MAPK依赖性途径来解释。早期研究提示蛋白激酶C（PKC）对sHSP磷酸化有作用，但后来基于报道的纯化PKC在体外磷酸化sHSP能力较差而受到质疑。本研究根据PKC家族的亚型复杂性重新评估了PKC在sHSP磷酸化中的作用。我们评估了从妊娠中期和晚期两个妊娠阶段获取的大鼠黄体中sHSP的磷酸化状态，这两个阶段表达不同水平的新型PKC亚型PKC-δ。二维蛋白质印迹分析表明，HSP-27在妊娠晚期黄体中的体内磷酸化程度更高，这与PKC-δ丰富且活跃的发育阶段相对应。妊娠晚期黄体提取物含有一种脂质敏感的HSP激酶活性，使用羟基磷灰石和S-Sepharose柱色谱法可将其与PKC-δ完全共纯化。为了确定是否可能存在特定PKC亚型对sHSP的优先磷酸化，对与在大鼠黄体中检测到的PKC亚型相对应的纯化重组PKC亚型进行了体外HSP激酶活性评估。重组PKC-δ在体外有效催化sHSP的磷酸化，PKC-α作为HSP激酶的有效性为PKC-δ的30 - 50%；测试的其他PKC（β1、β2、ε和ζ）是较差的HSP激酶。这些结果表明，特定的PKC家族成员在体外可作为直接的HSP激酶。此外，在妊娠晚期PKC-δ丰富且活跃时观察到黄体中HSP-27磷酸化增强，这表明特定的PKC家族成员在体内对sHSP磷酸化事件有贡献。
