👤 Danaé S Larsen

The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C-induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis. Show less

Mutations in the MYBPC3 gene, encoding the sarcomere protein myosin-binding protein C, are among the most frequent causes of autosomal dominant familial hypertrophic cardiomyopathy (FHC). We studied the frequency, type, and pathogenetic mechanism of MYBPC3 mutations in an unselected cohort of 81 FHC families, consecutively enrolled at a tertiary referral center. Nine mutations, six of which were novel, were found in 10 (12.3%) of the families using single-strand conformation polymorphism and DNA sequencing. A frameshift mutation in exon 2 clearly suggests that haploinsufficiency is a pathogenetic mechanism in FHC. In addition, splice site mutations in exon 6 and intron 31, a deletion in exon 13, and a nonsense mutation in exon 25, all lead to premature termination codons, most likely causing loss of function and haploinsufficiency. Furthermore, there were two missense mutations (D228N and A833 T) and one in-frame deletion (DeltaLys813). A considerable intrafamilial variation in phenotypic expression of MYBPC3-based FHC was noted, and we suggest that mutations influencing stability of mRNA could play a role in the variable penetrance and expressivity of the disease, perhaps via partial haploinsuffciency. Show less

Limited proteolysis of carboxypeptidase A from bovine pancreas with subtilisin Carlsberg generates a stable intermediate, carboxypeptidase S, whose esterase and peptidase activities are increased and decreased, respectively, under standard assay conditions. Carboxypeptidase S was isolated by affinity chromatography. Sequence analysis shows that it is cleaved solely at the Ala154-Gly155 bond. Its enzymatic properties were determined under stopped-flow conditions with Dns-Gly-Ala-Phe and its ester analogue Dns-Gly-Ala-OPhe. For both substrates, the Km values are increased 30-40-fold. The kcat value for peptide hydrolysis is virtually unaffected whereas that for ester hydrolysis is increased 10-fold. The magnitude of the Km effect is equivalent to a loss of 9 kJ/mol of binding energy and likely reflects a disruption of the network of hydrogen bonds that links Tyr-248 and Arg-145 to the backbone carbonyls of Ala-154 and Gly-155. The difference in kcat effects for the two substrate classes is related to differences in the chemical nature of the rate-determining step. Product release is rate determining for catalytic hydrolysis of ester substrates, and hence, the increase in kcat indicates that dissociation of products is facilitated as a result of the Ala154-Gly155 bond scission. The changes in enzymatic activity accompanying limited proteolysis are due to conformational alterations in the vicinity of the active center of the molecule. The affinity of a monoclonal antibody, mAb 100, directed toward the antigenic determinant located between residues 209 and 218 in carboxypeptidase A is diminished considerably for carboxypeptidase S.(ABSTRACT TRUNCATED AT 250 WORDS) Show less