Chellaiah Meenakshi A, Kuppuswamy Dhandapani, Lasky Larry, Linder Stefan
Department of Biomedical Sciences, University of Maryland Dental School, Baltimore, Maryland 21201.
Gazes Cardiac Research Institute, Cardiology Division, Medical University of South Carolina, Charleston, South Carolina 29425.
J Biol Chem. 2007 Mar 30;282(13):10104-10116. doi: 10.1074/jbc.M608957200. Epub 2007 Feb 5.
The activities of different kinases have been correlated to the phosphorylation of Wiscott-Aldrich syndrome protein (WASP) by studies in multiple cell systems. The purpose of this study was to elucidate the regulatory mechanisms involved in WASP phosphorylation and the resulting sealing ring formation in osteoclasts. The phosphorylation state of WASP and WASP-interacting proteins was determined in osteoclasts treated with osteopontin or expressing either constitutively active or kinase-defective Src by adenovirus-mediated delivery. In vitro kinase analysis of WASP immunoprecipitates exhibited phosphorylation of c-Src, PYK2, WASP, protein-tyrosine phosphatase (PTP)-PEST, and Pro-Ser-Thr phosphatase-interacting protein (PSTPIP). Phosphorylation of these proteins was increased in osteopontin-treated and constitutively active Src-expressing osteoclasts. Pulldown analysis with glutathione S-transferase-fused proline-rich regions of PTP-PEST revealed coprecipitation of WASP, PYK2, c-Src, and PSTPIP proteins with the N-terminal region (amino acids 294-497) of PTP-PEST. Similarly, interaction of the same signaling proteins, as well as PTP-PEST, was observed with glutathione S-transferase-fused proline-rich regions of WASP. Furthermore, osteopontin stimulation or constitutively active Src expression resulted in serine phosphorylation and inhibition of WASP-associated PTP-PEST. The inhibition of PTP-PEST was accompanied by an increase in tyrosine phosphorylation of WASP and other associated signaling proteins. Experiments with an inhibitor to phosphatase and small interference RNA to PTP-PEST confirmed the involvement of PTP-PEST in sealing ring formation and bone resorption. WASP, which is identified in the sealing ring of resorbing osteoclasts, also demonstrates colocalization with c-Src, PYK2, PSTPIP, and PTP-PEST in immunostaining analyses. Our findings suggest that both tyrosine kinase(s) and the tyrosine phosphatase PTP-PEST coordinate the formation of the sealing ring and thus the bone-resorbing function of osteoclasts.
通过在多个细胞系统中的研究，不同激酶的活性已与威斯科特-奥尔德里奇综合征蛋白（WASP）的磷酸化相关联。本研究的目的是阐明破骨细胞中WASP磷酸化及由此产生的密封环形成所涉及的调节机制。在用骨桥蛋白处理或通过腺病毒介导递送表达组成型活性或激酶缺陷型Src的破骨细胞中，测定WASP及与WASP相互作用蛋白的磷酸化状态。对WASP免疫沉淀物进行的体外激酶分析显示，c-Src、PYK2、WASP、蛋白酪氨酸磷酸酶（PTP）-PEST和脯氨酸-丝氨酸-苏氨酸磷酸酶相互作用蛋白（PSTPIP）发生了磷酸化。在骨桥蛋白处理的和表达组成型活性Src的破骨细胞中，这些蛋白的磷酸化增加。用谷胱甘肽S-转移酶融合的PTP-PEST富含脯氨酸区域进行的下拉分析显示，WASP、PYK2、c-Src和PSTPIP蛋白与PTP-PEST的N端区域（氨基酸294-497）共沉淀。同样，用谷胱甘肽S-转移酶融合的WASP富含脯氨酸区域也观察到相同信号蛋白以及PTP-PEST的相互作用。此外，骨桥蛋白刺激或组成型活性Src表达导致WASP相关的PTP-PEST的丝氨酸磷酸化和抑制。PTP-PEST的抑制伴随着WASP和其他相关信号蛋白酪氨酸磷酸化的增加。用磷酸酶抑制剂和针对PTP-PEST的小干扰RNA进行的实验证实了PTP-PEST参与密封环形成和骨吸收。在吸收性破骨细胞的密封环中鉴定出的WASP，在免疫染色分析中也显示与c-Src、PYK2、PSTPIP和PTP-PEST共定位。我们的研究结果表明，酪氨酸激酶和酪氨酸磷酸酶PTP-PEST共同协调密封环的形成，从而协调破骨细胞的骨吸收功能。
