Abstract
Phostensin is encoded by KIAA1949. 5′-RACEanalysis has been used to identify the translation start site of phostensin mRNA, indicating that it encodes 165 amino acids with an apparent molecular weight of 26 kDa on SDS-PAGE. This low-molecular-weight phostensin is present in human peripheral blood mononuclear cells and many leukemic cell lines. Phostensin is a protein phosphatase-1(PP1) binding protein. It also contains one actin-binding motif at its C-terminal region and binds to the pointed ends of actin filaments, modulating actin dynamics. In the current study, a high-molecular-weight phostensin is identified by using immunoprecipitationin combination with a proteomic approach. This new species of phostensin is also encoded by KIAA1949 and consists of 613 amino acids with an apparent molecular weight of 110 kDa on SDS-PAGE. The low-molecular-weight and high-molecular-weight phostensins were named as phostensin-α and phostensin-β, respectively. Although phostensin-α is the C-terminal region of phostensin-β, it is not degraded from phostensin-β. Phostensin-β is capable of associating with PP1 and actin filaments, and is present in many cell lines.
Highlights
Phostensin, a protein phosphatase-1 F-actin cytoskeleton targeting subunit, is encoded by KIAA1949 [1,2,3]
Cellular proteins were extracted by 1% SDS and ultrasonication. 5 and 10 μg of crude proteins extracted from HeLa and Jurkat cells, respectively, were analyzed by SDS-PAGE (10%), followed by western blotting using the PT2 monoclonal antibody; (C) Phostensin-β is present in many cell lines
PP1 and actin were precipitated by PT-2 monoclonal antibody from protein extracts of Jurkat cells, it may arise from interaction with phostensin-α instead of phostensin-β (Figure 1A)
Summary
Phostensin, a protein phosphatase-1 F-actin cytoskeleton targeting subunit, is encoded by KIAA1949 [1,2,3]. The open reading frame of KIAA1949 was predicted to encode a putative protein of 613 amino acids by the GENSCAN program [2,3] This full-length transcript of KIAA1949 gene was not observed by the early 5'-RACE analysis that only identified a shorter transcript encoding a smaller protein of 165 amino acids with an apparent molecular weight of 26 kDa on SDS-PAGE [1]. The sequence of this short-length protein was the C-terminal region of the predicted full-length protein. We apply co-immunoprecipitationin combination with proteomic approach to analyze the crude protein extracts of Jurkat cells and identify a high-molecular-weight species of phostensin encoded by the full-length transcript of KIAA1949 gene with an apparent molecular weight of 110 kDa on SDS-PAGE
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