Abstract
The microtubule organizing centre (MTOC) or the centrosome serves a crucial role in the establishment of cellular polarity, organization of interphase microtubules and the formation of the bipolar mitotic spindle. We have elucidated the genomic structure of a gene encoding the sarcolemmal membrane-associated protein (SLMAP), which encodes a 91 kDa polypeptide with a previously uncharacterized N-terminal sequence encompassing a forkhead-associated (FHA) domain that resides at the centrosome. Anti-peptide antibodies directed against SLMAP N-terminal sequences showed colocalization with gamma-tubulin at the centrosomes at all phases of the cell cycle. Agents that specifically disrupt microtubules did not affect SLMAP association with centrosomes. Furthermore, SLMAP sequences directed a reporter green fluorescent protein (GFP) to the centrosome, and deletions of the newly identified N-terminal sequence from SLMAP prevented the centrosomal targeting. Deletion-mutant analysis concluded that overall, structural determinants in SLMAP were responsible for centrosomal targeting. Elevated levels of centrosomal SLMAP were found to be lethal, whereas mutants that lacked centrosomal targeting inhibited cell growth accompanied by an accumulation of cells at the G2/M phase of the cell cycle.
Highlights
The microtubule organizing centre (MTOC) of the cell is the centrosome, a complex organelle that fulfills multiple functions including the nucleation of interphase microtubules, the establishment of cellular polarity, the formation and positioning of the bipolar mitotic spindle, and the segregation of chromosomes
Genomic organization of the sarcolemmal membrane-associated protein (SLMAP) gene We previously reported that the 3′ region of the SLMAP gene is composed of 11 exons and encodes a 37 kDa SLMAP polypeptide, which is expressed in a tissue-specific manner (Wielowieyski et al, 2000)
Intron sizes varied from 87 bp to over 50.5 kb of genomic DNA, whereas SLMAP exons ranged in size from 51 bp to over 1.8 kb (Table 3)
Summary
The microtubule organizing centre (MTOC) of the cell is the centrosome, a complex organelle that fulfills multiple functions including the nucleation of interphase microtubules, the establishment of cellular polarity, the formation and positioning of the bipolar mitotic spindle, and the segregation of chromosomes (reviewed by Doxsey, 2001; Nigg, 2002). In view of the pivotal role of the MTOC in ensuring genomic stability, various kinases and phosphatases anchored at centrosomes function as central regulators of centrosome activity (Meraldi et al, 1999; Hinchcliffe et al, 1999; Sluder and Hinchcliffe, 2000; Nigg, 2002). Examples include p34cdc, cAMP-dependent kinase II (PKA), Polo kinase, Cdc14A phosphatase, STK15(BTAK) and Nek kinase (reviewed by Mayor et al, 1999; Meraldi and Nigg, 2002; Mailand et al, 2002; Whitehead and Salisbury, 1999). While the functional roles of many centrosomal kinases and phosphatases continue to be further defined, the repertoire of centrosome-associated substrates remains to be uncovered
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