Regulation of protein kinase C Apl II by serotonin receptors in Aplysia.

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.

Structural basis of substrate recognition and specificity in the N-end rule pathway.

The N-end rule links the half-life of a protein to the identity of its N-terminal residue. Destabilizing N-terminal residues are recognized by E3 ubiquitin ligases, termed N-recognins. A conserved structural domain called the UBR box is responsible for their specificity. Here we report the crystal structures of the UBR boxes of the human N-recognins UBR1 and UBR2, alone and in complex with an N-end rule peptide, Arg-Ile-Phe-Ser. These structures show that the UBR box adopts a previously undescribed fold stabilized through the binding of three zinc ions to form a binding pocket for type 1 N-degrons. NMR experiments reveal a preference for N-terminal arginine. Peptide binding is abrogated by N-terminal acetylation of the peptide or loss of the positive charge of the N-terminal residue. These results rationalize and refine the empirical rules for the classification of type 1 N-degrons. We also confirm that a missense mutation in UBR1 that is responsible for Johanson-Blizzard syndrome leads to UBR box unfolding and loss of function.