Spatial Distribution of Protein Kinase A Activity during Cell Migration Is Mediated by A-kinase Anchoring Protein AKAP Lbc

Abstract

Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility.

Integrin α6β4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation

“…In our recent analysis of transcriptional changes mediated by integrin ␣ 6 ␤ 4 in these cells, we find that AKAP-Lbc mRNA expression is upregulated in MDA/␤ 4 cells 2.5-fold over control cells (4). To determine whether AKAPLbc is the Rho GEF responsible for RhoA activation in the MDA/␤ 4 transfectants, we reduced AKAP-Lbc by two separate siRNAs as done previously (39), plated cells on collagen coated dishes, stimulated cells with 100 nM LPA, and then assessed cell lysates for RhoA activity and AKAP-Lbc expression. As shown in Fig.…”

“…Suspended cells (3 ϫ 10 6 ) were electroporated with 200 nM control small interfering (si)RNA (nontargeting; Dharmacon) or individual siRNAs specific for AKAP-Lbc (AKAP13) as reported previously (39). Individual sequences for AKAP-Lbc are 5=-UCAACAGACUCAC-UAAAUAUU-3= (number 3) and 5=-GGAAGAAGCUUGUACGU-GAUU-3= (number 5).…”

Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium

“…PKA is required for the starvation-induced aggregation of Dictyostelium cells (Mann and Firtel, 1991;Mann et al, 1997), a process driven by chemotaxis, and was found to be involved in controlling the directional extension of pseudopods during migration (Stepanovic et al, 2005;Zhang et al, 2003). In mammalian cells, PKA has been shown to play a central role in actin-based cell migration through the differential regulation of Rac, Rho and Rap1 GTPases, as well as of VASP, PI3K, PAK and LIM kinases, at the leading edge of migrating cells (Chen et al, 2005;Howe, 2004;Howe et al, 2005;Jones and Sharief, 2005;Lim et al, 2008;Nadella et al, 2009;Paulucci-Holthauzen et al, 2009;Takahashi et al, 2013;Toriyama et al, 2012;Zimmerman et al, 2013).…”

WAVE2 Forms a Complex with PKA and Is Involved in PKA Enhancement of Membrane Protrusions

“…These results suggest that WAVE2 makes a complex with PKA and acts as AKAP that recruits PKA at membrane protrusions. In addition to WAVE2, integrin ␣4 (37) and AKAP-lbc (38) have been reported to form a complex with PKA and recruit PKA to protrusions in colon carcinoma cells and CHO cells. Interestingly, AKAP-lbc ablation reduces but does not completely disrupt PKA localization at protrusions (38).…”