Abstract
Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA.
Highlights
Tecnologia, Mexico). 3 To whom correspondence should be addressed: Dept. of Pharmacology, tion, adhesion, and morphogenesis
PKA Interacts with P-Rex1—To identify proteins with affinity for P-Rex1, a G␥- and phosphatidylinositol-3,4,5-trisphosphate-dependent RacGEF involved in cell migration [28], we used P-Rex1-PDZ-PDZ domains as bait in a yeast two-hybrid screen with a human endothelial cDNA library
We found that in yeast P-Rex1-PDZ-PDZ interacted with the C-terminal region of type I-␣ PKA regulatory subunit (PRKAR1a), including CNB B, the second cAMP binding domain
Summary
Tecnologia, Mexico). 3 To whom correspondence should be addressed: Dept. of Pharmacology, tion, adhesion, and morphogenesis.
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