Protein kinases transduce signals to regulate a wide array of cellular functions in eukaryotes. A generalizable method for optical control of kinases would enable fine spatiotemporal interrogation or manipulation of these various functions. We report the design and application of single-chain cofactor-free kinases with photoswitchable activity. We engineered a dimeric protein, pdDronpa, that dissociates in cyan light and reassociates in violet light. Attaching two pdDronpa domains at rationally selected locations in the kinase domain, we created the photoswitchable kinases psRaf1, psMEK1, psMEK2, and psCDK5.
During mitosis, each duplicated chromosome must be accurately attached to the microtubule spindle, which pulls the chromosomes to opposite poles of the cell, where they are segregated to daughter cells. A number of mitotic kinases orchestrate mitosis to ensure accurate segregation, including cyclin-dependent kinase 1 (CDK1), the Polo-like kinases, and the Aurora kinases (1). The kinase ATR (ataxia telangiectasia and Rad3-related), which is involved in DNA damage responses during interphase of the cell cycle, has also been shown to prevent chromosome segregation errors (2). However, this role of ATR was presumed to be an indirect effect. On page 108 of this issue, Kabeche et al. (3) unveil a mitosis-specific ATR activity that ensures proper chromosome segregation and that this activity is dependent on a specific three-stranded nucleic acid structure known as an R loop.
Hormones can transmit signals through adenosine 3ʹ,5ʹ-monophosphate (cAMP) to precise intracellular locations. The fidelity of these responses relies on the activation of localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory type II (RII) subunits with A-kinase–anchoring proteins (AKAPs) confers location, and catalytic (C) subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that AKAP79 constrains RII-C subassemblies within 150 to 250 angstroms of its targets. Native mass spectrometry established that these macromolecular assemblies incorporated stoichiometric amounts of cAMP.
When the rice blast fungus enters a rice cell, the plasma membrane stays intact, so the rice cell remains viable. The fungus then moves to adjacent cells via plasmodesmata, the plant's intercellular channels. Sakulkoo et al. used a chemical genetic approach to selectively inhibit a single MAP (mitogen-activated protein) kinase, Pmk1, in the blast fungus. Inhibition of Pmk1 trapped the fungus within a rice cell. Pmk1 regulated the expression of a suite of effector genes involved in suppression of host immunity, allowing the fungus to manipulate plasmodesmal conductance.
With early life likely to have existed in a hot environment, enzymes had to cope with an inherent drop in catalytic speed caused by lowered temperature. Here we characterize the molecular mechanisms underlying thermoadaptation of enzyme catalysis in adenylate kinase using ancestral sequence reconstruction spanning 3 billion years of evolution. We show that evolution solved the enzyme's key kinetic obstacle--how to maintain catalytic speed on a cooler Earth--by exploiting transition-state heat capacity. Tracing the evolution of enzyme activity and stability from the hot-start toward modern hyperthermophilic, mesophilic, and psychrophilic organisms illustrates active pressure versus passive drift in evolution on a molecular level, refutes the debated activity/stability trade-off, and suggests that the catalytic speed of adenylate kinase is an evolutionary driver for organismal fitness.