Ditlevsen Dorte Kornerup, Owczarek Sylwia, Berezin Vladimir, Bock Elisabeth
Protein Laboratory, Department of Neuroscience and Pharmacology, University of Copenhagen, Panum Institute, Building 6.2, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.
Neurochem Int. 2008 Nov;53(5):137-47. doi: 10.1016/j.neuint.2008.06.011. Epub 2008 Jul 4.
Although a large number of signalling cascades are known to be activated downstream of NCAM, only little is known regarding the hierarchical relationship between the involved molecules in the individual cascades and the level of cross talk between the cascades. Here, we evaluated the requirement of putative upstream signalling cascades for the phosphorylation of the kinases extracellular signal-regulated kinase (ERK) and Akt and the transcription factor cyclic adenosine monophosphate (cAMP) response-element binding protein (CREB) following stimulation of NCAM in rat cerebellar granule neurons with an NCAM ligand, the C3d peptide. NCAM-mediated ERK phosphorylation depended on activation of the fibroblast growth factor receptor (FGFR), Src-family kinases, MEK (MAP and ERK kinase) and G(0)/G(i)-proteins, whereas NCAM-mediated CREB phosphorylation depended on the activity of Src-family kinases and MEK. NCAM-specific Akt phosphorylation depended on cyclic guanosine monophosphate (cGMP) and phosphatidylinositide 3-kinase (PI3K). All three phosphorylation events were independent of activation of the signalling molecules phospholipase C, protein kinase C, protein kinase A, and CamKII, which all have been demonstrated previously to be involved in NCAM signalling. For comparison, we also evaluated the role of upstream signalling cascades on fibroblast growth factor 2 (FGF2)-mediated phosphorylation of ERK, Akt, and CREB and found that FGF2 required the activity of both FGFR and Src-family kinases for phosphorylation of ERK, Akt, and CREB. MEK was required for phosphorylation of ERK and CREB, but not Akt, whereas G(0)/G(i)-proteins were necessary for phosphorylation of Akt and CREB, and cGMP was necessary for Akt phosphorylation. We thus demonstrate that even though NCAM and FGF2 have many signalling features in common, and even though both are known to activate FGFR, there are a number of differences in the intracellular signalling network activated by the NCAM ligand C3d and the FGFR ligand FGF2.
尽管已知大量信号级联反应在神经细胞黏附分子（NCAM）下游被激活，但对于各个级联反应中所涉及分子之间的层级关系以及级联反应之间的相互作用程度却知之甚少。在此，我们评估了假定的上游信号级联反应对于用NCAM配体C3d肽刺激大鼠小脑颗粒神经元后，细胞外信号调节激酶（ERK）和Akt激酶以及转录因子环磷酸腺苷（cAMP）反应元件结合蛋白（CREB）磷酸化的必要性。NCAM介导的ERK磷酸化依赖于成纤维细胞生长因子受体（FGFR）、Src家族激酶、MEK（丝裂原活化蛋白激酶和细胞外信号调节激酶激酶）和G(0)/G(i)蛋白的激活，而NCAM介导的CREB磷酸化依赖于Src家族激酶和MEK的活性。NCAM特异性的Akt磷酸化依赖于环磷酸鸟苷（cGMP）和磷脂酰肌醇3激酶（PI3K）。所有这三种磷酸化事件均独立于信号分子磷脂酶C、蛋白激酶C、蛋白激酶A和钙/钙调蛋白依赖性蛋白激酶II（CamKII）的激活，而这些分子先前均已被证明参与NCAM信号传导。为作比较，我们还评估了上游信号级联反应对成纤维细胞生长因子2（FGF2）介导的ERK、Akt和CREB磷酸化的作用，发现FGF2使ERK、Akt和CREB磷酸化需要FGFR和Src家族激酶的活性。MEK是ERK和CREB磷酸化所必需的，但不是Akt磷酸化所必需的，而G(0)/G(i)蛋白是Akt和CREB磷酸化所必需的，cGMP是Akt磷酸化所必需的。因此，我们证明，尽管NCAM和FGF2有许多共同的信号特征，并且两者均已知可激活FGFR，但由NCAM配体C3d和FGFR配体FGF2激活的细胞内信号网络存在许多差异。
