ABSTRACT
Precise cell division is essential for multicellular development, and defects in this process have been linked to cancer. Septins are a family of proteins that are required for mammalian cell division, but their function and mode of regulation during this process are poorly understood. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) phosphorylates septin 9 (SEPT9) upon mitotic entry, and this phosphorylation controls association with the proline isomerase, Pin1. Both SEPT9 and Pin1 are critical for mediating the final separation of daughter cells. Expression of mutant SEPT9 that is defective in Pin1 binding was unable to rescue cytokinesis defects caused by SEPT9 depletion but rather induced dominant-negative defects in cytokinesis. However, unlike SEPT9 depletion, Pin1 was not required for the accumulation of the exocyst complex at the midbody. These results suggest that SEPT9 plays multiple roles in abscission, one of which is regulated by the action of Cdk1 and Pin1.
Subject(s)
CDC2 Protein Kinase/metabolism , Cytokinesis/physiology , Peptidylprolyl Isomerase/metabolism , Septins/metabolism , CDC2 Protein Kinase/genetics , Gene Expression Regulation/physiology , HeLa Cells , Humans , Mutation , NIMA-Interacting Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/genetics , Phosphorylation/physiology , Protein Binding , Septins/geneticsABSTRACT
Septins are a highly conserved family of GTP-binding cytoskeletal proteins implicated in multiple cellular functions, including membrane transport, apoptosis, cell polarity, cell cycle regulation, cytokinesis, and oncogenesis. Here we describe the characterization of a novel interacting partner of the septin family, initially cloned from a human testis expression library following yeast two-hybrid isolation to identify SEPT9 binding partners. Upon further genomic characterization and bioinformatics analyses it was determined that this novel septin-interacting partner was also a new member of the mammalian septin family, named SEPT14. SEPT14 maps to 7p11.2 in humans and includes a conserved GTPase domain and a predicted carboxy-terminus coiled-coil domain characteristic of other septins. Three potential translational start methionines were identified by 5' RACE-PCR encoding proteins of 432-, 427-, and 425-residue peptides, respectively. SEPT14 shares closest homology to SEPT10, a human dendritic septin, and limited homology to SEPT9 isoforms. SEPT14 colocalized with SEPT9 when coexpressed in cell lines, and epitope-tagged forms of these proteins coimmunoprecipitated. Moreover, SEPT14 was coimmunoprecipitated from rat testes using SEPT9 antibodies, and yeast two-hybrid analysis suggested SEPT14 interactions with nine additional septins. Multitissue Northern blotting showed testis-specific expression of a single 5.0-kb SEPT14 transcript. RT-PCR analysis revealed that SEPT14 was not detectable in normal or cancerous ovarian, breast, prostate, bladder, or kidney cell lines and was only faintly detected in fetal liver, tonsil, and thymus samples. Interestingly, SEPT14 was expressed in testis but not testicular cancer cell lines by RT-PCR, suggesting that further investigation of SEPT14 as a testis-specific tumor suppressor is necessary.
Subject(s)
Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Testis/metabolism , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 7 , Cloning, Molecular , Cytoskeletal Proteins/chemistry , DNA/genetics , GTP-Binding Proteins/chemistry , Humans , Male , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Protein Structure, Tertiary , Rats , Septins , Sequence Homology, Amino Acid , Tissue Distribution , Two-Hybrid System TechniquesABSTRACT
Since their essential role in cytokinesis was first shown in yeast, the septins have been described to function in diverse cellular contexts. The members of this unique class of GTPases are capable of binding and hydrolyzing GTP, associating with membranes and oligomerizing into higher order structures. Here we describe Sept12, a novel septin, identified in a yeast two hybrid screen using Sept5 as the bait. Sept12 contains the primary sequence elements of a septin and is capable of interacting with other septins. In addition, Sept12 purifies with bound nucleotide and binds to phosphoinositides, confirming its identity as a septin. RT-PCR and Northern blots reveal that Sept12 mRNA is expressed predominantly in testis, and this is supported by tissue Western blots. In rats, Sept12 protein levels rise upon sexual maturity and the Sept12 protein colocalizes with the annulus in isolated mature spermatozoa. Further, coexpression of Sept12 with Sept4, an essential annulus component, results in complete colocalization of both proteins into robust and highly curved filaments in CHO cells. This study suggests Sept12 may be involved in mammalian fertility.
Subject(s)
GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , Spermatozoa/metabolism , Amino Acid Sequence , Animals , Cricetinae , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/isolation & purification , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Male , Molecular Sequence Data , Rats , Rats, Wistar , Septins , Sperm Tail/metabolism , Spermatozoa/cytologyABSTRACT
Septins are a highly conserved family of filamentous GTPases required for cell division in a wide range of eukaryotic organisms. In a recent issue of Nature, Vrabioiu and Mitchison use polarized fluorescence microscopy to show that septin filaments undergo a highly orchestrated rotation, from a longitudinal to circumferential orientation, coincident with splitting of the septin ring during cytokinesis (Vrabioiu and Mitchison, 2006).
