Abstract
Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.
Highlights
Production of monoclonal antibodies against Tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) By immunizing recombinant mouse TMAP/CKAP2 protein from bacterial over-expression, two monoclonal antibodies against mTMAP/ CKAP2, B-1-13 and D-12-3, were produced
It has been previously reported that mTMAP/CKAP2 protein always appears as double bands regardless of the cell cycle phase, whereas the human counterpart appears as a single band (Bae et al, 2003; Hong et al, 2007), and the cause of such difference is currently unknown
We have previously observed a mitosis-specific shift/retardation of the TMAP/CKAP2 protein band on a SDS-PAGE gel (Hong et al, 2007), which suggests that TMAP/CKAP2 is modified by phosphorylation at the M phase
Summary
Tumor associated microtubule associated protein (TMAP), known as cytoskeleton associated protein 2 (CKAP2) is frequently upregulated in various malignancies, including gastric adenocarcinoma, diffuse B-cell lymphoma and cutaneous T-cell lymphoma (Maouche-Chretien et al, 1998; Eichmuller et al, 2001; Bae et al, 2003), and detected in various cancer cell lines (Bae et al, 2003; Jin et al, 2004). The exact cellular functions of TMAP/CKAP2 remain unknown. TMAP/CKAP2 is primarily localized to microtubules and centrosomes during interphase and to mitotic spindles and spindle poles during mitosis (Maouche-Chretien et al, 1998; Bae et al, 2003; Jin et al, 2004; Hong et al, 2007). While the level of TMAP/CKAP2 expression is low or undetectable in growth-arrested primary HFFs and NIH 3T3 cells, its expression starts to incline as cells enter the cell cycle and peaks at G2/M phases (Jeon et al, 2006). Recent reports have shown that TMAP/CKAP2 has microtubule-stabilizing properties and may contribute
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