RESUMEN
The His-x-Asp (HxD) motif is one of the most conserved structural components of the catalytic core of protein kinases; however, the functional role of the conserved histidine is unclear. Here we report that replacement of the HxD-histidine with Arginine or Phenylalanine in Aurora A abolishes both the catalytic activity and auto-phosphorylation, whereas the Histidine-to-tyrosine impairs the catalytic activity without affecting its auto-phosphorylation. Comparisons of the crystal structures of wild-type (WT) and mutant Aurora A demonstrate that the impairment of the kinase activity is accounted for by (1) disruption of the regulatory spine in the His-to-Arg mutant, and (2) change in the geometry of backbones of the Asp-Phe-Gly (DFG) motif and the DFG-1 residue in the His-to-Tyr mutant. In addition, bioinformatics analyses show that the HxD-histidine is a mutational hotspot in tumor tissues. Moreover, the H174R mutation of the HxD-histidine, in the tumor suppressor LKB1 abrogates the inhibition of anchorage-independent growth of A549 cells by WT LKB1. Based on these data, we propose that the HxD-histidine is involved in a conserved inflexible organization of the catalytic core that is required for the kinase activity. Mutation of the HxD-histidine may also be involved in the pathogenesis of some diseases including cancer.
Asunto(s)
Dominio Catalítico , Secuencia Conservada , Histidina/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Secuencias de Aminoácidos , Arginina/genética , Aurora Quinasa A/química , Aurora Quinasa A/genética , Línea Celular Tumoral , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutación/genética , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Relación Estructura-Actividad , Tirosina/genética , Difracción de Rayos XRESUMEN
The formation of new genes is a primary driving force of evolution in all organisms. The de novo evolution of new genes from non-protein-coding genomic regions is emerging as an important additional mechanism for novel gene creation. Y chromosomes underlie sex determination in mammals and contain genes that are required for male-specific functions. In this study, a search was undertaken for Y chromosome de novo genes derived from non-protein-coding sequences. The Y chromosome orphan gene variable charge, Y-linked (VCY)2, is an autosome-derived gene that has sequence similarity to large autosomal fragments but lacks an autosomal protein-coding homolog. VCY2 locates in the amplicon containing long DNA fragments that were transposed from autosomes to the Y chromosome before the ape-monkey split. We confirmed that VCY2 cannot be encoded by autosomes due to the presence of multiple disablers that disrupt the open reading frame, such as the absence of start or stop codons and the presence of premature stop codons. Similar observations have been made for homologs in the autosomes of the chimpanzee, gorilla, rhesus macaque, baboon and out-group marmoset, which suggests that there was a non-protein-coding ancestral VCY2 that was common to apes and monkeys that predated the transposition event. Furthermore, while protein-coding orthologs are absent, a putative non-protein-coding VCY2 with conserved disablers was identified in the rhesus macaque Y chromosome male-specific region. This finding implies that VCY2 might have not acquired its protein-coding ability before the ape-monkey split. VCY2 encodes a testis-specific expressed protein and is involved in the pathologic process of male infertility, and the acquisition of this gene might improve male fertility. This is the first evidence that de novo genes can be generated from transposed autosomal non-protein-coding segments, and this evidence provides novel insights into the evolutionary history of the Y chromosome.
Asunto(s)
Evolución Molecular , Proteínas/genética , Testículo/metabolismo , Cromosoma Y/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Pan troglodytes , Filogenia , Homología de Secuencia de Ácido NucleicoRESUMEN
Microtubule affinity-regulating kinase 2 (MARK2)/PAR-1b and protein kinase A (PKA) are both involved in the regulation of microtubule stability and neurite outgrowth, but whether a direct cross-talk exists between them remains unclear. Here, we found the disruption of microtubule and neurite outgrowth induced by MARK2 overexpression was blocked by active PKA. The interaction between PKA and MARK2 was confirmed by coimmunoprecipitation and immunocytochemistry both in vitro and in vivo. PKA was found to inhibit MARK2 kinase activity by phosphorylating a novel site, serine 409. PKA could not reverse the microtubule disruption effect induced by a serine 409 to alanine (Ala) mutant of MARK2 (MARK2 S409A). In contrast, mutation of MARK2 serine 409 to glutamic acid (Glu) (MARK2 S409E) did not affect microtubule stability and neurite outgrowth. We propose that PKA functions as an upstream inhibitor of MARK2 in regulating microtubule stability and neurite outgrowth by directly interacting and phosphorylating MARK2.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Microtúbulos/metabolismo , Neuritas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/metabolismo , Animales , Células HEK293 , Humanos , Fosforilación , RatasRESUMEN
AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK alpha1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha1(394) (alpha1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha1(394), alpha1(335), alpha2(398), and even heterotrimer alpha1beta1gamma1. Based on PT1-docked AMPK alpha1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.