We purified microtubules from a mammalian mitotic extract and obtained an amino acid sequence from each microtubule-associated protein by using mass spectrometry. Most of these proteins are known spindle-associated components with essential functional roles in spindle organization. We generated antibodies against a protein identified in this collection and refer to it as astrin because of its association with astral microtubule arrays assembled in vitro. Astrin is Ϸ134 kDa, and except for a large predicted coiled-coil domain in its C-terminal region it lacks any known functional motifs. Astrin associates with spindle microtubules as early as prophase where it concentrates at spindle poles. It localizes throughout the spindle in metaphase and anaphase and associates with midzone microtubules in anaphase and telophase. Astrin also localizes to kinetochores but only on those chromosomes that have congressed. Deletion analysis indicates that astrin's primary spindle-targeting domain is at the C terminus, although a secondary domain in the N terminus can target some of the protein to spindle poles. Thus, we have generated a comprehensive list of major mitotic microtubule-associated proteins, among which is astrin, a nonmotor spindle protein.T he spindle is a complex microtubule-based superstructure responsible for chromosome segregation in mitosis and meiosis (1-6). Spindle microtubules are organized in a complex process that requires both motor and nonmotor microtubuleassociated proteins. A great deal of emphasis has been placed on the functional contribution that motor proteins make to spindle organization because of their inherent biological activity. The strong sequence homology of the conserved motor domain in these proteins has facilitated both their rapid identification and characterization during spindle assembly. These investigations have demonstrated that motors of both the kinesin and dynein families play important roles in such varied aspects of spindle organization as spindle bipolarity (6), chromosome movement (7-10), checkpoint regulation (11, 12), spindle pole organization (13-16), and the regulation of microtubule dynamics (17). In contrast, the functional contribution that nonmotor proteins make to spindle organization has not progressed as rapidly. In part, the lag in progress in defining the roles of nonmotor spindle proteins stems from the lack of clearly defined biological activity of nonmotor proteins. A further complication arises from the lack of any clear homology among nonmotor proteins to facilitate their identification. Thus, although some nonmotor spindle proteins have been characterized (18,19), others are less well understood (20,21), and many others may await identification.In this article we use mass spectrometry to identify the proteins associated with microtubules assembled in a cell-free mitotic extract under conditions where Ͼ90% of microtubules are organized into astral arrays. Most proteins identified through this strategy have known functional roles for spindle assembly. A pro...
The hallmark of the 8p12 stem cell myeloproliferative disorder (MPD) is the disruption of the FGFR1 gene, which encodes a tyrosine kinase receptor for members of the fibroblast growth factor family.FGFR1 can be fused to at least 3 partner genes at chromosomal regions 6q27, 9q33, or 13q12. We report here the cloning of the t(8;9)(p12;q33) and the detection of a novel fusion betweenFGFR1 and the CEP110 gene, which codes for a novel centrosome-associated protein with a unique cell-cycle distribution. CEP110 is widely expressed at various levels in different tissues and is predicted to encode a 994-amino acid coiled-coil protein with 4 consensus leucine zippers [L-X(6)-L-X(6)-L-X(6)-L]. Both reciprocal fusion transcripts are expressed in the patient's cells. The CEP110-FGFR1 fusion protein encodes an aberrant tyrosine kinase of circa 150-kd, which retains most of CEP110 with the leucine zipper motifs and the catalytic domain of FGFR1. Transient expression studies show that the CEP110-FGFR1 protein has a constitutive kinase activity and is located within the cell cytoplasm.
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