Abstract
Many newly synthesized cellular proteins pass through the Golgi complex from where secretory transport carriers sort them to the plasma membrane and the extracellular environment. The formation of these secretory carriers at the trans-Golgi network is promoted by the protein kinase D (PKD) family of serine/threonine kinases. Here, using mathematical modeling and experimental validation of the PKD activation and substrate phosphorylation kinetics, we reveal that the expression level of the PKD substrate deleted in liver cancer 1 (DLC1), a Rho GTPase-activating protein that is inhibited by PKD-mediated phosphorylation, determines PKD activity at the Golgi membranes. RNAi-mediated depletion of DLC1 reduced PKD activity in a Rho-Rho-associated protein kinase (ROCK)-dependent manner, impaired the exocytosis of the cargo protein horseradish peroxidase, and was associated with the accumulation of the small GTPase RAB6 on Golgi membranes, indicating a protein-trafficking defect. In summary, our findings reveal that DLC1 maintains basal activation of PKD at the Golgi and Golgi secretory activity, in part by down-regulating Rho-ROCK signaling. We propose that PKD senses cytoskeletal changes downstream of DLC1 to coordinate Rho signaling with Golgi secretory function.
Highlights
Many newly synthesized cellular proteins pass through the Golgi complex from where secretory transport carriers sort them to the plasma membrane and the extracellular environment
To address whether protein kinase D (PKD) activity might be subject to Rho-dependent feedback regulation, we first examined whether the expression of active RhoA leads to PKD activation and concomitant inhibitory phosphorylation of the RhoGAP deleted in liver cancer 1 (DLC1), a direct
We used a combination of experiments on the PKD regulation network and mathematical modeling of phosphorylation dynamics to describe the molecular interactions among PKD, Rho, and DLC1
Summary
Dept. of Dermatology, University of Tubingen, 72076 Tubingen, Germany. Rho proteins are ubiquitously expressed small GTPases that coordinate actin and microtubule cytoskeleton rearrangements, thereby regulating diverse cellular processes such as cell adhesion and migration, cell division, and membrane trafficking [9]. Upon PKD-mediated phosphorylation, DLC1 is bound and sequestered by 14-3-3 proteins, thereby preventing it from inactivating Rho-GTP These observations raise the question of whether a positive feedback involving Rho GTPase signaling exists, maintaining cellular PKD activity and Golgi secretory function. We tested this hypothesis by a data-driven modeling approach that captures PKD activation depending on DLC1mediated Rho regulation. Our model anticipates a DLC1-dependent negative effect of Rho signaling on PKD activity These predictions were confirmed in subsequent cellular experiments, which further uncovered a novel role for DLC1 in the regulation of protein secretion from TGN membranes
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