ABSTRACTTransmembrane Ser/Thr kinases containing extracellular PASTA (penicillin-binding protein [PBP]andSer/Thr-associated) domains are ubiquitous amongActinobacteriaandFirmicutesspecies. Such PASTA kinases regulate critical bacterial processes, including antibiotic resistance, cell division, cell envelope homeostasis, and virulence, and are sometimes essential for viability. Previous studies of purified PASTA kinase fragments revealed they are capable of autophosphorylationin vitro, typically at multiple sites on the kinase domain. Autophosphorylation of a specific structural element of the kinase known as the activation loop is thought to enhance kinase activity in response to stimuli. However, the role of kinase phosphorylation at other sites is largely unknown. Moreover, the mechanisms by which PASTA kinases are deactivated once their stimulus has diminished are poorly understood.Enterococcus faecalisis a Gram-positive intestinal bacterium and a major antibiotic-resistant opportunistic pathogen. InE. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, and such antimicrobials trigger enhanced phosphorylation of IreKin vivo. Here we identify multiple sites of phosphorylation on IreK and evaluate their functionin vivoandin vitro. While phosphorylation of the IreK activation loop is required for kinase activity, we found that phosphorylation at a site distinct from the activation loop reciprocally modulates IreK activityin vivo, leading to diminished activity (and diminished antimicrobial resistance). Moreover, this site is important for deactivation of IreKin vivoupon removal of an activating stimulus. Our results are consistent with a model in which phosphorylation of IreK at distinct sites reciprocally regulates IreK activityin vivoto promote adaptation to cell wall stresses.IMPORTANCETransmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous amongActinobacteriaandFirmicutesspecies and regulate critical processes, including antibiotic resistance, cell division, and cell envelope homeostasis. Previous studies of PASTA kinase fragments revealed autophosphorylation at multiple sites. However, the functional role of autophosphorylation and the relative impacts of phosphorylation at distinct sites are poorly understood. The PASTA kinase ofEnterococcus faecalis, IreK, regulates intrinsic resistance to antimicrobials. Here we identify multiple sites of phosphorylation on IreK and show that modification of IreK at distinct sites reciprocally regulates IreK activity and antimicrobial resistancein vivo. Thus, these results provide new insights into the mechanisms by which PASTA kinases can regulate critical physiological processes in a wide variety of bacterial species.