The complicated and entangled cell signaling network is dynamically regulated by a wide array of enzymes such as kinases. It remains desirable but challenging to specifically modulate individual, endogenous kinases within a cell, particularly in a spatial-temporally controlled fashion. Current strategies toward regulating the intracellular functions of a kinase of interest either lack specificity or require genetic engineering that may perturb its physiological activity. Herein, we harnessed a bacterial effector OspF for optical and chemical modulation of the endogenous mitogen-activated protein kinase (MAPK) cascade in living cells and mice. The phospho-lyase OspF provided high specificity and spatial resolution toward the desired kinase such as the extracellular signal-regulated kinase (ERK), while the genetically encoded bioorthogonal decaging strategy enabled its temporal activation in living systems. The photocaged OspF (OspF*) was applied to dissect the subcellular signaling roles of ERK in nucleus as opposed to cytoplasm, while the chemically caged OspF (OspFc) was introduced into living mice to modulate ERK-mediated gene expression. Finally, our spatially and chemically controlled OspFc was further used to precisely tune immune responses in T cells. Together, our bioorthogonal engineering strategy on bacterial effectors offers a general tool to modulate cell signaling with high specificity and spatial-temporal resolution.