Schematic representation of the human CMS and protein sequence. (A) Alignment of human cDNA clones isolated in the yeast two-hybrid screen, 5′ rapid amplification of complementary DNA ends (RACE) and λgt11 cDNA library screen with respect to the full-length CMS cDNA. PR, proline-rich region. (B) Predicted protein sequence of CMS. Sequence numbers are shown on the left. The SH3 domains are underlined. Proline-rich sequences are marked in bold, the CC domain is marked in italics, and the putative actin binding sites are bold underlined. IP, immunoprecipitation. (C) In vitro interaction of p130^Cas and CMS. 293T cells were transiently transfected with the flag-tagged CMS yeast TH clone (CMS-TH) together with the CasSH3 domain or full-length Cas (both GST tagged) or the vector alone (GST control). Cell lysates were immunoprecipitated with anti-flag antibody or glutathione Sepharose beads, and precipitates were subjected to SDS/PAGE and probed with anti-GST antibody and anti-flag antibody, respectively. The upper blot was striped and reprobed with anti-flag antibody. (D) Direct interaction of CMS with p130^Cas. Five hundred micrograms of protein from 293T cells expressing the various CMS peptides or C3G used as a positive control was immunoprecipitated with the antibodies indicated above. Blots were probed for binding with ³²P-labeled GST-p130^CasSH3 domain.

Tissue distribution of CMS mRNA. (A) Northern blot analysis of poly(A)⁺ RNA from adult human tissues by using the ³²P-labeled CMS cDNA clone isolated in the two-hybrid screen. Hybridization analysis of the same Northern blot by using a ³²P-labeled β-actin cDNA probe (Lower). (B) Relative expression levels of CMS in different human tissues and developmental stages. Adult tissue: A1, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hippocampus; A8, medulla oblongata; B1, occipital lobe; B2, putamen; B3, substantia nigra; B4, temporal lobe; B5, thalamus; B6, nucleus accumbeus; B7, spinal cord; C1, heart; C2, aorta; C3, skeletal muscle; C4, colon; C5, bladder; C6, uterus; C7, prostate; C8, stomach; D1, testis; D2, ovary, D3, pancreas, D4, pituitary gland; D5, adrenal gland; D6, thyroid gland; D7, salivary gland; D8, mammary gland; E1, kidney; E2, liver; E3, small intestine; E4, spleen; E5, thymus; E6, peripheral leukocyte; E7, lymph node; E8, bone marrow; F1, appendix; F2, lung; F3, trachea; F4, placenta. Fetal tissue: G1, brain; G2, heart; G3, kidney; G4, liver; G5, spleen; G6, thymus; G7, lung. Controls (100 ng each): H1, yeast total RNA; H2, yeast tRNA; H3, Escherichia coli rRNA; H4, E. coli DNA; H5, poly(rA); H6, human C₀t 1 DNA; H7, human DNA; H8, human DNA (500 ng).

CMS is localized in the cytoplasm and colocalizes with actin and p130^Cas to membrane ruffles. (A and B) COS-7 cells grown in medium containing 10% FBS, plated on poly-d-lysine coverslips, were transfected with the flag-tagged CMS constructs CMS, CMS/TH, and CMS/3SH3 as indicated. Cells were fixed and stained with rhodamine-labeled phalloidin (lane 1) or with anti-flag antibody (lanes 3 and 4). Merged images are shown in lane 2. Actin clustering adjacent to the nucleus (B, lane 1 marked with an arrow). Vesicles in CMS cells are indicated by arrows (B, lane 3). (C and D) Membrane ruffle induction in COS-7 cells transfected with flag-tagged CMS or GST-tagged p130^Cas. Serum-starved cells were treated with 10 nM phorbol 12-myristate 13-acetate (PMA) for 10 min, fixed, and subjected to immunofluorescence analysis by using the anti-flag or p130^Cas antibody (lane 3). Actin filaments were visualized by staining with rhodamine-labeled phalloidin (lane 1). The cells were analyzed by confocal microscopy (×250 and ×630). Yellow-orange color in merged image shows colocalization of CMS and p130^Cas with actin (lane 2, marked with arrows). (Bar, 20 μm.)

Analysis of SH3-containing proteins for their interaction with CMS. (A) GST-fusion peptides coupled to glutathione Sepharose beads were incubated with lysates of 293T cells expressing flag-tagged CMS. Precipitates were analyzed by SDS/PAGE and WB with anti-flag antibody. (B) In vivo interaction of CMS with the tyrosine kinases Fyn and Yes. Lysates of 293T cells expressing flag-tagged CMS-TH or control vector were incubated with anti-Fyn and anti-Yes antibody coupled to protein A/G agarose beads. Immunoprecipitates (IP) were analyzed in SDS/PAGE and WB with anti-flag antibody. (C) Kinase activity is associated with CMS. Lysates of 293T cells expressing flag-tagged CMS or vector control were incubated with anti-flag antibody coupled to protein A/G agarose, and immunoprecipitates (IP) were subjected to a kinase assay. (D) Tyrosine phosphorylation of CMS. 293T cell lysates expressing flag-tagged CMS together with the indicated tyrosine kinases [wild type (WT) or kinase dead (KD)] were immunoprecipitated (IP) with antiphoshotyrosine antibody (4G10) and analyzed by WB with antiphosphotyrosine antibody and anti-CMS polyclonal serum. Total cell lysates were analyzed for expression of the transfected plasmids (not shown).

Dimerization of CMS is mediated through the COOH-terminal CC region. (A) Helical wheel representation of the putative leucine zipper in the CC region of CMS. (B) 293T cells were cotransfected with either empty vectors, GST vector, and flag-tagged CMS-TH (a), FLAG vector and GST-tagged CMS/P-CC (b), or CMS-TH and CMS/P-CC. Immunoprecipitates (IP) were analyzed by WB using anti-flag antibody (Upper). The expression levels were analyzed in total cell lysates by WB. (C) Mapping of the dimerization site in CMS. CMS target constructs for expression in yeast were introduced in vector pACT2 and transfected together with the bait CMS/P-CC cloned in pAS2–1. Transformants were grown on plates lacking leucine and tryptophane. Three single colonies were analyzed for interaction in a β-galactosidose filter assay (19). −, no interaction. +, interaction.