Moreno-García Miguel E, Sommer Karen, Haftmann Claudia, Sontheimer Clayton, Andrews Sarah F, Rawlings David J
Center for Immunity and Immunotherapies, Seattle Children's Research Institute, University of Washington School of Medicine, Seattle, WA 98109, USA.
J Immunol. 2009 Dec 1;183(11):7362-70. doi: 10.4049/jimmunol.0902438. Epub 2009 Nov 16.
Phosphorylation of CARMA1 is a crucial event initiating the assembly of IkappaB kinase and JNK signaling complexes downstream of activated Ag receptors. We previously mapped three protein kinase C (PKC) target sites in murine CARMA1 in vitro, and demonstrated that mutation of two of these serines (S564 and S657) resulted in reduced NF-kappaB activation, whereas mutation of the third serine (S649) had no clear effect. In this study, we report that when low concentrations of Ag receptor activators are used, loss of S649 (by mutation to alanine) promotes enhanced IkappaB kinase and JNK activation in both B and T cell lines. Reconstitution of CARMA1(-/-) DT40 B cells with CARMA1 S649A leads to increased cell death and reduced cell growth in comparison to wild-type CARMA1, likely a result of enhanced JNK activation. To directly determine whether S649 is modified in vivo, we generated phospho-specific Abs recognizing phospho-S649, and phospho-S657 as a positive control. Although phospho-S657 peaked and declined rapidly after Ag receptor stimulation, phospho-S649 occurred later and was maintained for a significantly longer period poststimulation in both B and T cells. Interestingly, phospho-S657 was completely abolished in PKCbeta-deficient B cells, whereas delayed phosphorylation at S649 was partially intact and depended, in part, upon novel PKC activity. Thus, distinct PKC-mediated CARMA1 phosphorylation events exert opposing effects on the activation status of CARMA1. We propose that early phosphorylation events at S657 and S564 promote the initial assembly of the CARMA1 signalosome, whereas later phosphorylation at S649 triggers CARMA1 down-regulation.
CARMA1的磷酸化是启动活化抗原受体下游IκB激酶和JNK信号复合物组装的关键事件。我们之前在体外确定了小鼠CARMA1中的三个蛋白激酶C（PKC）靶位点，并证明其中两个丝氨酸（S564和S657）的突变导致NF-κB激活降低，而第三个丝氨酸（S649）的突变没有明显影响。在本研究中，我们报道当使用低浓度的抗原受体激活剂时，S649缺失（突变为丙氨酸）会促进B细胞和T细胞系中IκB激酶和JNK的激活增强。与野生型CARMA1相比，用CARMA1 S649A重建CARMA1(-/-) DT40 B细胞会导致细胞死亡增加和细胞生长减少，这可能是JNK激活增强的结果。为了直接确定S649在体内是否被修饰，我们生成了识别磷酸化S649的磷酸特异性抗体，并将磷酸化S657作为阳性对照。尽管磷酸化S657在抗原受体刺激后迅速达到峰值并下降，但磷酸化S649出现较晚，并且在B细胞和T细胞刺激后维持的时间明显更长。有趣的是，PKCβ缺陷的B细胞中磷酸化S657完全消失，而S649的延迟磷酸化部分完整，并且部分依赖于新型PKC活性。因此，不同的PKC介导的CARMA1磷酸化事件对CARMA1的激活状态产生相反的影响。我们提出，S657和S564的早期磷酸化事件促进了CARMA1信号小体的初始组装，而S649的后期磷酸化触发了CARMA1的下调。
