p21-activated kinase (PAK)-interacting exchange factor (PIX) is known to be involved in regulation of Cdc42/Rac GTPases and PAK activity. PIX binds to the proline-rich region of PAK, and regulates biological events through activation of Cdc42/Rac GTPase. To further investigate the role of PIX we produced monoclonal antibodies (Mab) against beta PAK. Three clones; N-C6 against N-terminal half and C-A3 and C-B7 against C- terminal half of beta PAK were generated and characterized. N-C6 Mab detected beta PAK as a major band in most cell lines. C-A3 Mab recognizes GIT-binding domain (GBD), but it does not interfere with GIT binding to beta PAK. Using C-A3 Mab possible beta PAK interaction with actin in PC12 cells was examined. beta PAK Mab (C-A3) specifically precipitated actin of the PC12 cell lysates whereas actin Mab failed to immunoprecpitate beta PAK. Co-sedimentation of PC12 cell lysates with the polymerized F-actin resulted in the recovery of most of beta PAK in the cell lysates. These results suggest that beta PAK may not interact with soluble actin but with polymerized F-actin and revealed that beta PAK constitutes a functional complex with actin. These data indicate real usefulness of the beta PAK Mab in the study of beta PAK role(s) in regulation of actin cyoskeleton.
Actins/*metabolism ; Animals ; Antibodies, Monoclonal/immunology ; Cell Cycle Proteins/immunology/metabolism/*physiology ; Cell Line, Tumor ; Cytoskeletal Proteins/metabolism ; Epitope Mapping ; Guanine Nucleotide Exchange Factors/immunology/metabolism/*physiology ; Immunoprecipitation ; Mice ; Microfilaments/*physiology ; Protein Structure, Tertiary ; Rats ; Research Support, Non-U.S. Gov't

BACKGROUNDSam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line.

METHODSBy using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometric analyses were performed to investigate the Sam68 functions.

RESULTSA slower growth rate (2.1 hours growth elongation) and longer S phase (1.7 hours elongation) was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells.

CONCLUSIONSThe proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; physiology ; Animals ; B-Lymphocytes ; cytology ; immunology ; physiology ; Binding Sites ; genetics ; Blotting, Western ; Cell Cycle ; physiology ; Cell Death ; physiology ; Cell Growth Processes ; drug effects ; physiology ; Cell Line, Tumor ; Culture Media, Serum-Free ; pharmacology ; Mutation ; genetics ; Phosphorylation ; Proline ; genetics ; RNA-Binding Proteins ; genetics ; metabolism ; physiology ; Receptors, Antigen, B-Cell ; immunology ; physiology ; Signal Transduction ; drug effects ; physiology ; Tyrosine ; metabolism

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.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal/metabolism/pharmacology ; Cell Cycle/physiology ; Cells, Cultured ; Cytoskeletal Proteins/chemistry/immunology/*metabolism/physiology ; Epitope Mapping ; Hela Cells ; Humans ; Mice ; Mitosis/*physiology ; Molecular Sequence Data ; Phosphorylation ; Protein-Serine-Threonine Kinases/*metabolism ; Sequence Homology, Amino Acid ; Threonine/metabolism