Regulation of mixed-lineage kinase activation in JNK-dependent morphogenesis

Abstract

SummaryNormal cells respond appropriately to various signals, while sustaining proper developmental programs and tissue homeostasis. Inappropriate signal reception, response or attenuation, can upset the normal balance of signaling within cells, leading to dysfunction or tissue malformation. To understand the molecular mechanisms that regulate protein-kinase-based signaling in the context of tissue morphogenesis, we analyzed the domain requirements of Drosophila Slpr, a mixed-lineage kinase (MLK), for Jun N-terminal kinase (JNK) signaling. The N-terminal half of Slpr is involved in regulated signaling whereas the C-terminal half promotes cortical protein localization. The SH3 domain negatively regulates Slpr activity consistent with autoinhibition via a conserved proline motif. Also, like many kinases, conserved residues in the activation segment of the catalytic domain regulate Slpr. Threonine 295, in particular, is essential for function. Slpr activation requires dual input from the MAP4K Misshapen (Msn), through its C-terminal regulatory domain, and the GTPase Rac, which both bind to the LZ-CRIB region of Slpr in vitro. Although Rac is sufficient to activate JNK signaling, our results indicate that there are Slpr-independent functions for Rac in dorsal closure. Finally, expression of various Slpr constructs alone or with upstream activators reveals a wide-ranging response at the cell and tissue level.

Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling inDrosophila

“…Using site-directed mutagenesis by PCR overlap extension (Ho et al 1989), the following chimeric constructs were created: the Slpr-Tak kinase swap, ST K , consists of Slpr aa 1-128, Tak1 aa 19-271, and Slpr aa 383-1148, in that order. The Slpr-Tak C-terminus swaps, ST Ct and S AAA T Ct , consist of Slpr aa 1-516, with either a wild-type kinase domain or with activation loop alanine mutations, respectively (Garlena et al 2010), followed directly by Tak1 aa 272-678. The alternate Tak-Slpr kinase swap chimeras, TS K and TS AAA , consist of Tak1 aa 1-18, Slpr wild-type or triple alanine mutant kinase domain aa 128-385, followed directly by Tak1 aa 272-678.…”

“…w 1118 was used as a control. For mutants and transgenics, Bloomington (BL) Stock Center numbers are given if appropriate: UAS-Slpr, UASSlpr AAA , and UAS-SKLC (Garlena et al 2010), slpr BS06 (Polaski et al 2006), Tak1 2 BL# 26272 (Vidal et al 2001), UAS-Tak1 and UAS-Tak1 K46R (Mihaly et al 2001), egr GS9830 (UAS-eiger) , puc E69 (puc-lacZ) (Ring and Martinez Arias 1993), and UAS-srcEGFP BL# 5432. For constructs under the control of UAS sequences, expression was regulated by the Gal4 transcription factor (Brand and Perrimon 1993).…”

“…Given that one of the assays used to monitor a requirement for Tak1 is based on dominant interference of endogenous activity, we also generated a kinase domain swap in Tak1, TS AAA , using a Slpr kinase domain mutated in the activation loop to prevent activating phosphorylation. Our previous work demonstrated that this combination of alanine mutations disrupts phosphorylation and renders Slpr nonfunctional due to its inability to activate downstream JNK signaling (Garlena et al 2010).…”

“…The ability of Slpr to localize to the cell cortex in embryonic epithelium is attributed to the C-terminal half of the protein, and though this activity was nonessential in mutant rescue experiments, it contributed to maximal Slpr function (Garlena et al 2010). The C terminus of the Tak1 protein harbors a putative regulatory domain identifiable by an island of sequence conservation among homologs (Takatsu et al 2000;Mihaly et al 2001).…”

“…These functional domains are followed by a long C-terminal region lacking notable domains but enriched in phosphorylation motifs thought to modulate protein function and/or localization (Vacratsis et al 2002). Multistep activation of MLKs by upstream signals involves GTPase binding, relief of autoinhibition, dimerization, and phosphorylation by MAP4K proteins (Bock et al 2000;Vacratsis and Gallo 2000;Zhang and Gallo 2001;Du et al 2005;Garlena et al 2010;Kant et al 2011).…”