Article

Signaling from the Secretory Granule to the Nucleus: Uhmk1 and PAM

Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
Molecular Endocrinology (Impact Factor: 4.2). 08/2010; 24(8):1543-58. DOI: 10.1210/me.2009-0381
Source: PubMed

ABSTRACT Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine alpha-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent.

1 Bookmark
 · 
97 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Proteins derived from the Kalrn gene, encoding 2 Rho guanine nucleotide exchange factor (GEF) domains, affect dendritic and axonal morphogenesis. The roles of endogenous Kalirin-9 (Kal9) and Kalirin-12 (Kal12), the Kalrn isoforms expressed before synaptogenesis, have not been studied in neurite growth and maturation during early development. The Caenorhabditis elegans and Drosophila melanogaster orthologues of Kalrn encode proteins equivalent to Kal9 but, lacking a kinase domain, neither organism expresses a protein equivalent to Kal12. Both in vivo and in vitro analyses of cortical neurons from total Kalrn knockout mice, lacking all major Kalirin isoforms, revealed a simplified dendritic arbor and reduced neurite length. Using isoform-specific shRNAs to reduce Kal9 or Kal12 expression in hippocampal cultures resulted in stunted dendritic outgrowth and branching in vitro, without affecting axonal polarity. Exposing hippocampal cultures to inhibitors of the first GEF domain of Kalirin (ITX3, Z62954982) blunted neurite outgrowth and branching, confirming its essential role, without altering the morphology of neurons not expressing Kalrn. In addition, exogenous expression of the active kinase domain unique to Kal12 increased neurite number and length, whereas that of the inactive kinase domain decreased neurite growth. Our results demonstrate that both endogenous Kal9 and endogenous Kal12 contribute to dendritic maturation in early development.
    Cerebral Cortex 08/2014; DOI:10.1093/cercor/bhu182 · 8.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The adaptor protein 1A complex (AP-1A) transports cargo between the trans-Golgi network (TGN) and endosomes. In professional secretory cells, AP-1A also retrieves material from immature secretory granules (SGs). The role of AP-1A in SG biogenesis was explored using AtT-20 corticotrope tumor cells expressing reduced levels of the AP-1A μ1A subunit. A two-fold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non-condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue-stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α-amidating monooxygenase-1 (PAM-1), integral membrane enzymes that enter immature SGs. The non-condensing SGs contained POMC products and PAM-1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM-1 into PHM was unaltered, but PHM basal secretion was increased in sh-μ1A PAM-1 cells. Despite lacking a canonical AP-1A binding motif, yeast two-hybrid studies demonstrated an interaction between the PAM-1 cytosolic domain and AP-1A. Co-immunoprecipitation experiments with PAM-1 mutants revealed an influence of the luminal domains of PAM-1 on this interaction. Thus, AP-1A is crucial for normal SG biogenesis, function and composition.
    Traffic 06/2014; DOI:10.1111/tra.12194 · 4.71 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of PAM, a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wildtype and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His-cluster largely eliminated its pH-sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in hEK-293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wildtype AtT-20 cells; secretory products no longer accumulated in the TGN and secretory granule exocytosis was more responsive to secretagogue.
    Journal of Biological Chemistry 03/2014; 289(18). DOI:10.1074/jbc.M113.545947 · 4.60 Impact Factor