Spatial targeting of type II protein kinase A to filopodia mediates the regulation of growth cone guidance by cAMP

Abstract

The second messenger cyclic adenosine monophosphate (cAMP) plays a pivotal role in axonal growth and guidance, but its downstream mechanisms remain elusive. In this study, we report that type II protein kinase A (PKA) is highly enriched in growth cone filopodia, and this spatial localization enables the coupling of cAMP signaling to its specific effectors to regulate guidance responses. Disrupting the localization of PKA to filopodia impairs cAMP-mediated growth cone attraction and prevents the switching of repulsive responses to attraction by elevated cAMP. Our data further show that PKA targets protein phosphatase-1 (PP1) through the phosphorylation of a regulatory protein inhibitor-1 (I-1) to promote growth cone attraction. Finally, we find that I-1 and PP1 mediate growth cone repulsion induced by myelin-associated glycoprotein. These findings demonstrate that the spatial localization of type II PKA to growth cone filopodia plays an important role in the regulation of growth cone motility and guidance by cAMP.

14-3-3 Proteins Regulate Protein Kinase A Activity to Modulate Growth Cone Turning Responses

“…We further examined HSV-R18-EGFP-or HSV-WLRL-EGFP-transduced growth cones for indications of PKA activity. PKA regulates growth cone turning in part through targeting the PKA substrate PP-1 inhibitory protein I-1 (Han et al, 2007). We find that an antibody that recognizes the phosphorylated form of I-1 robustly stains a subset of neurites in P5 rat DRG explants.…”

“…cAMP binding to the regulatory subunits causes the release of the catalytic subunits and subsequent activation of PKA. We asked whether 14-3-3 isoforms could interact with PKA regulatory subunits ␣ or ␤ (RII ␣ and RII ␤) that are known to be present in growth cones (Han et al, 2007). First, we performed coimmunoprecipitation experiments in HEK 293T cotransfected with myc-tagged 14-3-3 isoforms and V5-tagged RII␣ or RII␤.…”

“…Introduction of R18 dissociates the PKA catalytic and regulatory subunits suggesting that 14-3-3 proteins stabilize the PKA holoenzyme. However, it is also well established that the spatial localization and regulation of PKA activity is essential for proper growth cone turning in Xenopus spinal neurons (Han et al, 2007) and it is conceivable that 14-3-3 proteins may have a parallel function cooperating with A-Kinase anchoring proteins (AKAPs) which anchor PKA to phosphorylation targets consequently regulating local PKA activity. Upon local stimulation of DRG growth cones with an NGF gradient, we do not find any evidence of asymmetric localization of 14-3-3␥ by immunofluorescence (data not shown).…”

“…Antagonizing cAMP in embryonic neurons can convert growth cone attraction to repulsion while elevating cAMP in postnatal neurons can convert repulsion to attraction and can promote CNS regeneration following injury (Neumann et al, 2002;Qiu et al, 2002). Both type II cAMP-dependent protein kinase A (PKA) and the exchange protein activated by cAMP (Epac) have been implicated as cAMP effectors affecting neurite outgrowth and growth cone turning (Cai et al, 2001;Han et al, 2007;Murray and Shewan, 2008) but the mechanisms regulating these effectors are unclear.…”

Calcium signaling in axon guidance

“…Other crucial components of this pathway include PKA, exchange protein directly activated by cAMP (Epac), inhibitor 1 (I1), and the second messenger cAMP. A recent mathematical model has proposed a mechanism for this molecular switch [42], based on a pathway uncovered by Wen et al [45] and Han et al [47] (Box 1 and Figure 2). Similar turning mechanisms have been proposed to underlie the response of various other cues, in particular morphogens [48,49].…”