Regulatory Mechanism Of The Synapses Of Amphids Defctive Kinase-B

The SAD(synapses of amphids defective)kinases,including SAD-A and SAD-B,belong to the AMP-activated protein kinase(AMPK)family.They play important roles in the regulation of neuronal development,including establishment of neuronal polarity,formation of terminal arbors,maturation of nerve terminals and release of neurotransmitter.SAD-B kinases also function in cell cycle progression,which is impotant in centrosome duplication.In addition,SAD-A is found to have a striking role in the energy metabolism in pancreas.The kinase activity of SAD is required for normal cell activity.Like other AMPK family members,SAD kinases contain a conserved kinase domain(KD),an adjacent ubiquitin-associated(UBA)domain,and a kinase associated1(KA1)domain.The recent study of mouse SAD-A identified a unique autoinhibitory sequence(AIS),which binds at the junction of the kinase domain(KD)and the ubiquitin-associated(UBA)domain and exerts autoregulation in cooperation with UBA.Here,we performed biochemical and structural experiments to investigate the regulatory mechanism of AIS in SAD-B kinase.Firstly,we studied biochemical characteristics of SAD-B KD-UBA(kinase domain and ubiquitin-associated domain): the result showed SAD-B could be readily phosphorylated at Thr189 by LKB1,and the kinetic parameters of fully phosphorylated SAD-B KD-UBA fragment(pKD-UBA)differs from that of SAD-A in our early studies.Secondly,we reported the crystal structure of the mouse SAD-B fragment including the AIS and the kinase associated domain 1(KA1)at 2.8 ? resolution.The KA1 domain is structurally conserved,while the isolated AIS sequence is highly flexible and solvent-accessible.Thirdly,our biochemical studies indicated that the SAD-B AIS exerted the same autoinhibitory role as that in SAD-A.The autoinhibition is conserved in SAD kinases.In conclusion,our studies reveal the autoinhibitory role of AIS in SAD-B kinase,and our results have contribution in elucidating the regulation of SAD kinase.