Compartmentalization of macromolecules is a ubiquitous molecular mechanism that drives numerous cellular functions. Appropriate organization of enzymes in space and time enables the precise transmission and integration of intracellular signals. Molecular scaffolds constrain signaling enzymes to influence the regional modulation of these physiological processes.  Mitochondrial targeting of protein kinases and protein phosphatases provides a means to locally control the phosphorylation status and action of proteins on the surface of this organelle. Dual-specificity A-kinase anchoring protein 1 (dAKAP1) is a multivalent binding protein that targets protein kinase A (PKA), RNAs and other signaling enzymes to the outer mitochondrial membrane. Many AKAPs recruit a diverse set of binding partners that coordinate a broad range of cellular process. Here, results of mass spectrometry and biochemical analyses reveal that dAKAP1 anchors additional components including the ribonucleoprotein granule components La-related protein 4 (LARP4) and polyadenylate-binding protein 1 (PABPC1). Local translation of mRNAs at organelles is a means to spatially control the synthesis of proteins. RNA-Seq data demonstrate that dAKAP1 binds mRNAs encoding proteins required for mitochondrial metabolism, including succinate dehydrogenase. Functional studies suggest that loss of dAKAP1-RNA interactions reduces mitochondrial electron transport chain activity. Hence, dAKAP1 plays a previously unappreciated role as a molecular interface between second messenger signaling and local protein synthesis machinery.

Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two proteinengineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1-and Src-PI3K-signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.any physiological and pathological processes are regulated by protein kinases. Therefore, dissection of kinase functions helps to uncover molecular mechanisms and guides the development of new therapeutic strategies (1). However, progress in our understanding of kinase-mediated signaling is often hampered by the limitations of available tools (2). Pharmacological inhibitors of kinases have been valuable for the study of kinase functions, but they often do not provide the desired specificity and they are not available for the majority of kinases (1, 3). Also, the application of inhibitors is limited to the identification of processes affected by the inactivation of the kinase. Activation of a kinase is often achieved by the treatment of cells with growth factors or other extracellular stimuli. However, this approach triggers a multitude of parallel signaling pathways, which significantly complicates the analysis of individual kinase functions (4). Specific activation or inactivation of a kinase can be achieved by genetic modifications. However, this method is susceptible to compensatory artifacts and does not allow us to control the level and timing of kinase activation. Thus, development of novel tools that combine high specificity and tight temporal control of kinase activity remains a necessity. Under physiological conditions, kinases are activated for a finite period, and the duration of this activation is critical for eliciting specific biological outcomes (5, 6). Therefore, to mimic the biological activity of a kinase we need to use methods that allow for transient activation of a kinase with precise temporal control. An optogenetic approach has been successfully used for transient regulation of Raf kinase by dimerization (7). However, this method relies on the fact that Raf dimerization is required for its activation and thus has limited applicability to other kinases. Therefore, development of broadly applicable methods for tightly contro...

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Highlights d SRC activation causes temporally distinct effects on the endothelial cell barrier d Initially, SRC causes endothelial barrier enhancement and VE cadherin rearrangement d VE cadherin phosphorylation on Y731 is required for SRCmediated barrier enhancement d Prolonged SRC activity cause barrier disruption