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.

Tyrosines outside the kinase core and dimerization are required for the mitogenic activity of RET/ptc2.

Defects in the c-ret proto-oncogene, a member of the protein tyrosine kinase receptor family, have recently been linked to two types of genetic syndromes, Hirschsprung's disease and the multiple endocrine neoplasia family of inherited cancers. RET/ptc2 is the product of a papillary thyroid carcinoma translocation event between the genes coding for c-ret and the type I alpha regulatory subunit of protein kinase A (RI alpha) (Lanzi, C., Borrello, M., Bongarzone, I., Migliazza, A., Fusco, A., Grieco, M., Santoro, M., Gambetta, R., Zunino, F., Della Porta, G., and Pierotti, M. (1992) Oncogene 7, 2189-2194). The resulting 596-residue protein contains the first two-thirds of RI alpha and the entire tyrosine kinase domain of c-ret (RETtk). An in vivo assay of growth stimulatory effects was developed, which consisted of microinjecting a RET/ptc2 expression plasmid into the nuclei of 10T1/2 mouse fibroblasts and observing the incorporation of 5-bromodeoxyuridine. This assay was used to determine that only the dimerization domain of RI alpha fused to RETtk is required for RET/ptc2's mitogenic activity. In addition, all of the reported Hirschsprung's disease point mutations in the RETtk (S289P, R421Q, and R496G) inactivate RET/ptc2 in our assay, confirming that these are loss of function mutations. Two tyrosines outside the conserved kinase core were also identified that are essential for full mitogenic activity of RET/ptc2. These two tyrosines, Tyr-350 and Tyr-586, are potential sites for Src homology 2 and phosphotyrosine binding domain interactions.

Identification of distinct domains for signaling and receptor interaction of the sensory rhodopsin I transducer, HtrI.

The phototaxis-deficient mutant of Halobacterium salinarium, Pho81, lacks both sensory rhodopsin I (SR-I) and its putative transducer protein HtrI, according to immunoblotting and spectroscopic criteria. From restriction analysis and selected DNA sequencing, we have determined that the SR-I- HtrI- phenotype results from an insertion of a 520-bp transposable element, ISH2, into the coding region of the SR-I apoprotein gene sopI and deletion of 11 kbp upstream of ISH2 including the first 164 bp of sopI and the entire htrI gene. SR-I and HtrI expression as well as full phototaxis sensitivity are restored by transformation with a halobacterial plasmid carrying the htrI-sopI gene pair and their upstream promoter region. An internal deletion of a portion of htrI encoding the putative methylation and signaling domains of HtrI (253 residues) prevents the restoration of phototaxis, providing further evidence for the role of HtrI as a transducer for SR-I. Analysis of flash-induced photochemical reactions of SR-I over a range of pH shows that the partially deleted HtrI maintains SR-I interactions sites responsible for modulation of the SR-I photocycle.

Primary structure of an archaebacterial transducer, a methyl-accepting protein associated with sensory rhodopsin I.

A methylated membrane protein of 97 kDa was suggested on the basis of mutant analysis to transduce signals from the phototaxis receptor sensory rhodopsin I to the flagellar motor in Halobacterium halobium. Here we report isolation of the proposed transducer protein, cloning of its gene based on partial protein sequences, the complete gene sequence, and analysis of the encoded primary structure. The 1611-base-pair gene termination codon overlaps the initiator ATG of the sopI gene, which encodes the sensory rhodopsin I apoprotein. The predicted size of 57 kDa for the methylated protein indicates an aberrant electrophoretic migration on SDS/polyacrylamide gels, as occurs with other acidic halophilic proteins. Putative promotor elements are located in an A+T-rich region upstream of the gene. Comparison of the translated nucleotide sequence with N-terminal sequence of the purified protein shows the protein is synthesized without a processed leader peptide and the N-terminal methionine is removed in the mature protein. The deduced protein sequence predicts two transmembrane helices near the N terminal that would anchor the protein to the membrane. Beyond this hydrophobic region of 46 residues, the remainder of the protein (536-amino acid residues total) is hydrophilic. The C-terminal 270 residues contain a region homologous to the signaling domains of eubacterial transducers (e.g., Escherichia coli Tsr protein), flanked by two regions homologous to the methylation domains of the transducer family. The protein differs from E. coli Tsr in that it does not have an extramembranous-receptor binding domain but instead has a more extended cytoplasmic region. Coexpression of the methyl-accepting protein gene (designated htrI) and sopI restores sensory rhodopsin I phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI region. These results extend the eubacterial transducer family to the archaebacteria and substantiate the proposal that the methylated membrane protein functions as a signal-transducing relay between sensory rhodopsin I and cytoplasmic sensory-pathway components.