PASTA kinase dimerization in Enterococcus faecalis does not require the extracellular PASTA domains

The penicillin- and Ser/Thr-kinase-associated (PASTA) kinase family of bacterial signaling proteins encompasses a widely conserved family of transmembrane Ser/Thr kinases defined by the presence of multiple PASTA domains comprising the extracellular module. PASTA kinases are found in nearly all gram-positive bacteria and are activated in response to cell wall stress to control critical physiological processes. A thorough understanding of the mechanisms of PASTA kinase signaling is important not just for greater insight into bacterial physiology but also to target PASTA kinases for therapeutic intervention. However, many fundamental features of the proposed model for signaling by PASTA kinases have not been tested using full-length kinase in cells of its native host under physiological conditions; hence, the mechanisms of PASTA kinase signaling have remained largely obscure. In this work, we tested fundamental features of the proposed model for PASTA kinase signaling using the representative PASTA kinase of Enterococcus faecalis , IreK. Through a combination of genetics and biochemistry, we established that IreK participates in self-association to form dimers in E. faecalis cells and that the self-associated form of IreK is functionally important for signaling. However, in contrast to the proposed model for PASTA kinase signaling, IreK dimerization does not require the extracellular PASTA module, and moreover, dimerization does not correlate with IreK signaling. Additionally, dimerization of IreK is not sufficient for kinase activation. Thus, regulation of PASTA kinase signaling in vivo is more complex than has been proposed.