PKA, PKC, and AKAP localization in and around the neuromuscular junction.
BACKGROUND
One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA) and protein kinase C (PKC). A-kinase anchoring proteins (AKAPs) can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIalpha in addition to the RII subunits.


RESULTS
Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIalpha at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ) and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIalpha and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIalpha and RIIbeta) are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCbeta appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane.


CONCLUSIONS
The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIalpha and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIalpha in these regions. Likewise, gravin may be an anchor of PKCbeta at the NMJ.
Perkins
G A
GA
Department of Neurosciences and the National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093-0608, USA. perkins@ncmir.ucsd.edu
Wang
L
L
Huang
L J
LJ
Humphries
K
K
Yao
V J
VJ
Martone
M
M
Deerinck
T J
TJ
Barraclough
D M
DM
Violin
J D
JD
Smith
D
D
Newton
A
A
Scott
J D
JD
Taylor
S S
SS
Ellisman
M H
MH
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis*; D002451:Cell Compartmentation / Q000502:physiology; D018797:Cell Cycle Proteins; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis*; D007150:Immunohistochemistry; D007366:Intercostal Muscles / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis; D008297:Male; D018613:Microscopy, Confocal; D009469:Neuromuscular Junction / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000096:biosynthesis*; D021122:Protein Subunits / Q000096:biosynthesis; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000096:biosynthesis; D013569:Synapses / Q000378:metabolism
D054758:A Kinase Anchor Proteins; C484639:AKAP10 protein, human; C491162:AKAP12 protein, human; D048868:Adaptor Proteins, Signal Transducing; C486333:Akap1 protein, mouse; C486332:Akap1 protein, rat; C491161:Akap12 protein, mouse; C102361:Akap12 protein, rat; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D007527:Isoenzymes; D021122:Protein Subunits; D011506:Proteins; D011978:Receptors, Nicotinic; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C