Townley Ian K, Schuyler Erin, Parker-Gür Michelle, Foltz Kathy R
Department of Molecular, Cellular and Developmental Biology and the Marine Science Institute, UC Santa Barbara Santa Barbara, CA 93106-9610, USA.
Dev Biol. 2009 Mar 15;327(2):465-77. doi: 10.1016/j.ydbio.2008.12.032. Epub 2009 Jan 3.
Egg activation at fertilization in deuterostomes requires a rise in intracellular Ca(2+), which is released from the egg's endoplasmic reticulum. In sea urchins, a Src Family Kinase (SpSFK1) is necessary for the PLCgamma-mediated signaling event that initiates this Ca(2+) release (Giusti, A.F., O'Neill, F.J., Yamasu, K., Foltz, K.R. and Jaffe, L.A., 2003. Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization. Dev. Biol. 256, 367-378.). Annotation of the Strongylocentrotus purpuratus genome sequence led to the identification of additional, predicted SFKs (Bradham, C.A., Foltz, D.R., Beane, W.S., Amone, M.I., Rizzo, F., Coffman, J.A., Mushegian, A., Goel, M., Morales, J., Geneviere, A.M., Lapraz, F., Robertson, A.J., Kelkar, H., Loza-Coll, M., Townley, I.K., Raisch, M., Roux, M.M., Lepage, T., Gache, C., McClay, D.R., Manning, G., 2006. The sea urchin kinome: a first look. Dev. Biol. 300, 180-193.; Roux, M.M., Townley, I.K., Raisch, M., Reade, A., Bradham, C., Humphreys, G., Gunaratne, H.J., Killian, C.E., Moy, G., Su, Y.H., Ettensohn, C.A., Wilt, F., Vacquier, V.D., Burke, R.D., Wessel, G. and Foltz, K.R., 2006. A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation. Dev. Biol. 300, 416-433.). Here, we describe the cloning and characterization of these 4 additional SFKs and test their function during the initial Ca(2+) release at fertilization using the dominant-interfering microinjection method coupled with Ca(2+) recording. While two of the new SFKs (SpFrk and SpSFK3) are necessary for Ca(2+) release, SpSFK5 appears dispensable for early egg to embryo transition events. Interestingly, SpSFK7 may be involved in preventing precocious release of Ca(2+). Binding studies indicate that only SpSFK1 is capable of direct interaction with PLCgamma. Immunolocalization studies suggest that one or more SpSFK and PLCgamma are localized to the egg cortex and at the site of sperm-egg interaction. Collectively, these data indicate that more than one SFK is involved in the Ca(2+) release pathway at fertilization.
后口动物受精时的卵子激活需要细胞内Ca(2+)浓度升高，Ca(2+)从卵子的内质网中释放出来。在海胆中，一种Src家族激酶（SpSFK1）对于磷脂酶Cγ（PLCγ）介导的启动这种Ca(2+)释放的信号转导事件是必需的（Giusti, A.F., O'Neill, F.J., Yamasu, K., Foltz, K.R.和Jaffe, L.A., 2003. 海胆卵子Src家族激酶在受精时启动Ca2+释放中的作用。《发育生物学》256, 367 - 378.）。对紫球海胆基因组序列的注释导致了其他预测的Src家族激酶的鉴定（Bradham, C.A., Foltz, D.R., Beane, W.S., Amone, M.I., Rizzo, F., Coffman, J.A., Mushegian, A., Goel, M., Morales, J., Geneviere, A.M., Lapraz, F., Robertson, A.J., Kelkar, H., Loza - Coll, M., Townley, I.K., Raisch, M., Roux, M.M., Lepage, T., Gache, C., McClay, D.R., Manning, G., 2006. 海胆激酶组：初步观察。《发育生物学》300, 180 - 193.；Roux, M.M., Townley, I.K., Raisch, M., Reade, A., Bradham, C., Humphreys, G., Gunaratne, H.J., Killian, C.E., Moy, G., Su, Y.H., Ettensohn, C.A., Wilt, F., Vacquier, V.D., Burke, R.D., Wessel, G.和Foltz, K.R., 2006. 海胆钙信号传导和卵子激活的功能基因组学和蛋白质组学视角。《发育生物学》300, 416 - 433.）。在这里，我们描述了这4种额外的Src家族激酶的克隆和特性，并使用显性干扰显微注射方法结合Ca(2+)记录来测试它们在受精时初始Ca(2+)释放过程中的功能。虽然其中两种新的Src家族激酶（SpFrk和SpSFK3）对于Ca(2+)释放是必需的，但SpSFK5对于早期卵子到胚胎的转变事件似乎是可有可无的。有趣的是，SpSFK7可能参与防止Ca(2+)过早释放。结合研究表明只有SpSFK1能够与PLCγ直接相互作用。免疫定位研究表明一种或多种Src家族激酶和PLCγ定位于卵子皮质以及精卵相互作用的部位。总的来说，这些数据表明不止一种Src家族激酶参与受精时的Ca(2+)释放途径。
