RESUMEN
The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We found that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 has diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Quinasas Similares a Doblecortina , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Microtúbulos/metabolismo , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Classical microtubule-associated proteins (MAPs) were originally identified based on their co-purification with microtubules assembled from mammalian brain lysate. They have since been found to perform a range of functions involved in regulating the dynamics of the microtubule cytoskeleton. Most of these MAPs play integral roles in microtubule organization during neuronal development, microtubule remodeling during neuronal activity, and microtubule stabilization during neuronal maintenance. As a result, mutations in MAPs contribute to neurodevelopmental disorders, psychiatric conditions, and neurodegenerative diseases. MAPs are post-translationally regulated by phosphorylation depending on developmental time point and cellular context. Phosphorylation can affect the microtubule affinity, cellular localization, or overall function of a particular MAP and can thus have profound implications for neuronal health. Here we review MAP1, MAP2, MAP4, MAP6, MAP7, MAP9, tau, and DCX, and how each is regulated by phosphorylation in neuronal physiology and disease. Developmental Dynamics 247:138-155, 2018. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Citoesqueleto/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Animales , Encéfalo/metabolismo , Humanos , FosforilaciónRESUMEN
Macroautophagy/autophagy is a dynamic and inducible catabolic process that responds to a variety of hormonal and environmental cues. Recent studies highlight the interplay of this central pathway in a variety of pathophysiological diseases. Although defective autophagy is implicated in melanocyte proliferation and pigmentary disorders, the mechanistic relationship between the 2 pathways has not been elucidated. In this study, we show that autophagic proteins LC3B and ATG4B mediate melanosome trafficking on cytoskeletal tracks. While studying melanogenesis, we observed spatial segregation of LC3B-labeled melanosomes with preferential absence at the dendritic ends of melanocytes. This LC3B labeling of melanosomes did not impact the steady-state levels of these organelles but instead facilitated their intracellular positioning. Melanosomes primarily traverse on microtubule and actin cytoskeletal tracks and our studies reveal that LC3B enables the assembly of microtubule translocon complex. At the microtubule-actin crossover junction, ATG4B detaches LC3B from melanosomal membranes by enzymatic delipidation. Further, by live-imaging we show that melanosomes transferred to keratinocytes lack melanocyte-specific LC3B. Our study thus elucidates a new role for autophagy proteins in directing melanosome movement and reveal the unconventional use of these proteins in cellular trafficking pathways. Such crosstalk between the central cellular function and housekeeping pathway may be a crucial mechanism to balance melanocyte bioenergetics and homeostasis.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Cisteína Endopeptidasas/metabolismo , Citoesqueleto/metabolismo , Melanosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Movimiento , Citoesqueleto de Actina/metabolismo , Animales , Citoesqueleto/ultraestructura , Dendritas/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Queratinocitos/metabolismo , Lípidos/química , Melanocitos/metabolismo , Melanocitos/ultraestructura , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Melanoma Experimental/ultraestructura , Melanosomas/ultraestructura , Ratones Endogámicos C57BL , Microtúbulos/metabolismo , PigmentaciónRESUMEN
Lipid modification of cytoplasmic proteins initiates membrane engagement that triggers diverse cellular processes. Despite the abundance of lipidated proteins in the human proteome, the key determinants underlying membrane recognition and insertion are poorly understood. Here, we define the course of spontaneous membrane insertion of LC3 protein modified with phosphatidylethanolamine using multiple coarse-grain simulations. The partitioning of the lipid anchor chains proceeds through a concerted process, with its two acyl chains inserting one after the other. Concurrently, a conformational rearrangement involving the α-helix III of LC3, especially in the three basic residues Lys65, Arg68, and Arg69, ensures stable insertion of the phosphatidylethanolamine anchor into membranes. Mutational studies validate the crucial role of these residues, and further live-cell imaging analysis shows a substantial reduction in the formation of autophagic vesicles for the mutant proteins. Our study captures the process of water-favored LC3 protein recruitment to the membrane and thus opens, to our knowledge, new avenues to explore the cellular dynamics underlying vesicular trafficking.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteínas Asociadas a Microtúbulos/química , Secuencia de Aminoácidos , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Membrana Dobles de Lípidos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , RatasRESUMEN
The recurrent interaction of skin with sunlight is an intrinsic constituent of human life, and exhibits both beneficial and detrimental effects. The apparent robust architectural framework of skin conceals remarkable mechanisms that operate at the interface between the surface and environment. In this Review, we discuss three distinct protective mechanisms and response pathways that safeguard skin from deleterious effects of ultraviolet (UV) radiation. The unique stratified epithelial architecture of human skin along with the antioxidant-response pathways constitutes the important defense mechanisms against UV radiation. The intricate pigmentary system and its intersection with the immune-system cytokine axis delicately balance tissue homeostasis. We discuss the relationship among these networks in the context of an unusual depigmenting disorder, vitiligo. The elaborate tunable mechanisms, elegant multilayered architecture and evolutionary selection pressures involved in skin and sunlight interaction makes this a compelling model to understand biological complexity.