Dissecting the function of Ack family kinases in Drosophila through understanding their interactions with Dock and Cdc42
Deregulation of Ack (Activated Cdc42-associated Kinase) family of non-receptor tyrosine kinases in mammals correlates with poor prognosis in cancers and has been implicated in promoting metastasis. In Drosophila, Ack family is constituted of two kinases: Ack and PR2. To further understand the in vivo function of this family we have conducted two projects: First we, we characterized the developmental defects of a null mutation in Drosophila Ack, which bears a high degree of sequence similarity to mammalian ACK1 but lacks a CRIB domain. We show that Ack, while not essential for viability, is critical for sperm formation. This function depends on Ack tyrosine kinase activity and is required cell autonomously in differentiating male germ cells at or after the spermatocyte stage. Ack associates predominantly with endocytic clathrin sites in spermatocytes, but disruption of Ack function has no apparent effect on clathrin localization and receptor-mediated internalization of Boss (Bride of sevenless) protein in eye discs. Instead, Ack is required for the subcellular distribution of Dock (dreadlocks), the Drosophila homolog of the SH2- and SH3-containing adapter protein Nck. Moreover, Dock forms a complex with Ack, and the localization of Dock in male germ cells depends on its SH2 domain. Together, our results suggest that Ack-dependent tyrosine phosphorylation recruits Dock to promote sperm differentiation. Second, despite the fact that ACK1 has been originally identified by its ability to bind to activated Cdc42, the physiological relevance of this binding has not been described. To test the significance of this interaction, we have established an over-expression system of various Cdc42 constructs in Drosophila germ cells. Similar to Drosophila embryos, expression of constitutively active Cdc42 (Cdc42G12V) in Drosophila spermatocytes results in cytokinesis defects, suggesting the step(s) affected by excessive Cdc42 activity is both meiotic and mitotic. Cdc42G12V is recruited to the site of cytokinesis ring constriction in germ cells where an increase in the level of tyrosine phosphorylation is also observed. This implies that a tyrosine kinase/substrate responds to Cdc42 activation. Indeed, loss-of-function mutations in PR2 but not Ack suppresses Cdc42G12V-dependent cytokinesis defect, suggesting that PR2 acts downstream of Cdc42. Moreover, PR2 is co-IPed with activated Cdc42 but not wild type Cdc42. Finally we show that Cdc42 G12V alters the subcellular localization of PR2 recruiting it to the site of cellular division.
Chang, Purdue University.
Molecular biology|Cellular biology|Developmental biology
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