Integration of cell cycle signals by multi-PAS domain kinases

Abstract

Spatial control of intracellular signaling relies on signaling proteins sensing their subcellular environment. In many cases, a large number of upstream signals are funneled to a master regulator of cellular behavior, but it remains unclear how individual proteins can rapidly integrate a complex array of signals within the appropriate spatial niche within the cell. As a model for how subcellular spatial information can control signaling activity, we have reconstituted the cell pole-specific control of the master regulator kinase/phosphatase CckA from the asymmetrically dividing bacterium Caulobacter crescentus. CckA is active as a kinase only when it accumulates within a microdomain at the new cell pole, where it co-localizes with the pseudokinase DivL. Both proteins contain multiple PAS domains, a multifunctional class of sensory domains present across the kingdoms of life. Here, we show that CckA uses its PAS domains to integrate information from DivL and on its own oligomerization state to control the balance of its kinase and phosphatase activities. We reconstituted the DivL-CckA complex on liposomes in vitro and found that DivL directly controls the CckA kinase-phosphatase switch, and that stimulation of either CckA catalytic activity depends on the second of its two PAS domains. We further show that CckA oligomerizes through a multi-domain interaction that is critical for stimulation of kinase activity by DivL, while DivL stimulation of CckA phosphatase activity is independent of CckA homo-oligomerization. Our results broadly demonstrate how signaling factors can leverage information from their subcellular niche to drive spatiotemporal control of cell signaling.\n\nSignificanceCells must constantly make decisions involving many pieces of information at a molecular level. Kinases containing multiple PAS sensory domains detect multiple signals to determine their signaling outputs. In the asymmetrically dividing bacterium Caulobacter crescentus, the multi-sensor proteins DivL and CckA promote different cell types depending upon their subcellular location. We reconstituted the DivL-CckA interaction in vitro and showed that specific PAS domains of each protein function to switch CckA between kinase and phosphatase activities, which reflects their functions in vivo. Within the context of the cell, our reconstitution illustrates how multi-sensor proteins can use their subcellular location to regulate their signaling functions.