Division of Bone and Mineral Research, Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts, USA; Division of Molecular Signaling and Biochemistry, Department of Health Improvement, Kyushu Dental University, Fukuoka, Japan.
Division of Bone and Mineral Research, Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts, USA; Division of Molecular Signaling and Biochemistry, Department of Health Improvement, Kyushu Dental University, Fukuoka, Japan.
J Biol Chem. 2021 Jan-Jun;296:100790. doi: 10.1016/j.jbc.2021.100790. Epub 2021 May 18.
Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into "stone." In contrast, deletion of the genes encoding other members of the Src family kinase (SFK) fails to produce an osteopetrotic phenotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other SFKs. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn, and Fyn were the most highly expressed SFKs in WT osteoclasts, whereas Hck, Lck, Blk, and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src-Hck and Src-Lyn constructs in which the unique, SH3, SH2, or catalytic domains had been swapped. We found that the Src unique, SH3, and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src's functionality is, in part, derived from an SH3-proximal proline-rich domain interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help clarify Src's unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.
c-Src 是一种广泛表达的酪氨酸激酶，如果将其删除，会导致破骨细胞功能障碍和骨质硬化（即“石骨症”）。相比之下，如果删除其他Src 家族激酶（SFK）的编码基因，则不会产生骨质硬化表型。这表明 c-Src 在破骨细胞中发挥着独特的功能，而其他 SFKs 无法替代。我们旨在确定 c-Src 在破骨细胞和骨吸收中发挥独特作用的分子基础。我们发现，c-Src、Lyn 和 Fyn 是 WT 破骨细胞中表达水平最高的 SFK，而 Hck、Lck、Blk 和 Fgr 的表达水平较低。破骨细胞中的足突带（一种独特的肌动蛋白组装簇）形成被破坏；引入组成型激活的 SFK 表明，只有 c-Src 和 Fyn 能够恢复这一过程。为了确定负责的关键结构域，我们构建了 Src-Hck 和 Src-Lyn 嵌合结构，其中交换了独特的 SH3、SH2 或催化结构域。我们发现，Src 的独特结构域、SH3 结构域和激酶结构域对于建立 Src 的功能都是至关重要的。但是，SH2 结构域可以被 Lyn 或 Hck 的 SH2 结构域取代。此外，我们证明 c-Src 的功能部分来源于与 c-Cbl 的 SH3 近端富含脯氨酸的结构域相互作用，导致 c-Cbl Tyr700 的磷酸化。这些数据有助于阐明 Src 在破骨细胞中细胞骨架组织中的独特功能，这是有效骨吸收所必需的，并解释了为什么 c-Src 不能被其他 SFK 替代。
