The product of the c-abl protooncogene is a nonreceptor tyrosine kinase found in both the cytoplasm and the nucleus. We report herein that cell adhesion regulates the kinase activity and subcellular localization of c-Abl. When fibroblastic cells are detached from the extracellular matrix, kinase activity of both cytoplasmic and nuclear c-Abl decreases, but there is no detectable alteration in the subcellular distribution. Upon adhesion to the extracellular matrix protein fibronectin, a transient recruitment of a subset of c-Abl to early focal contacts is observed coincident with the export of c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool of c-Abl is reactivated within 5 min of adhesion, but the nuclear c-Abl is reactivated after 30 min, correlating closely with its return to the nucleus and suggesting that the active nuclear c-Abl originates in the cytoplasm. In quiescent cells where nuclear c-Abl activity is low, the cytoplasmic c-Abl is similarly regulated by adhesion but the nuclear c-Abl is not activated upon cell attachment. These results show that c-Abl activation requires cell adhesion and that this tyrosine kinase can transmit integrin signals to the nucleus where it may function to integrate adhesion and cell cycle signals.Integrins are heterodimeric transmembrane receptors composed of an ␣ subunit and a ␤ subunit that bind extracellular matrix proteins such as fibronectin (FN) or other cell surface molecules such as the cell adhesion molecules VCAM-1 and ICAM-1 (for review, see ref. 1). Integrin cytoplasmic domains connect to the cytoskeleton to regulate cell spreading, cell migration, and cytoskeletal organization. Integrin-dependent signals are also critical for the adhesion-dependent regulation of gene expression, progression through the cell cycle, and differentiation. These biological effects are mediated through the activation of multiple signal transducers by cell adhesion, in particular tyrosine kinases such as focal adhesion kinase (2) and Src family members (3, 4), mitogen-activated protein (MAP) kinases (5, 6), protein kinase C (7, 8), phosphatidylinositol kinases (9), and the small GTPase Rho (10).The tyrosine kinase encoded by the c-abl protooncogene is found in both the cytoplasm and the nucleus (for review, see ref. 11). It is a multidomain protein containing src homology SH2 and SH3 domains, a tyrosine kinase domain, and binding domains for actin and DNA. The kinase activity of nuclear c-Abl is regulated during cell cycle progression. In quiescent and G 1 cells, nuclear c-Abl is kept in an inactive state by the retinoblastoma protein (RB) that binds to the c-Abl tyrosine kinase domain and inhibits its activity (12, 13). Phosphorylation of RB by cyclin-dependent kinases at the G 1 ͞S boundary disrupts the RB-c-Abl complex, leading to activation of the c-Abl tyrosine kinase. Activated nuclear c-Abl can phosphorylate the C-terminal repeated domain (CTD) of RNA polymerase II to modulate transcription (14, 15). Nuclear c-Abl is part of the RB-E2F complex (16); thus, t...

The ubiquitously expressed nonreceptor tyrosine kinase c-Abl contains three nuclear localization signals, however, it is found in both the nucleus and the cytoplasm of proliferating fibroblasts. A rapid and transient loss of c-Abl from the nucleus is observed upon the initial adhesion of fibroblasts onto a fibronectin matrix, suggesting the possibility of nuclear export [Lewis, J., Baskaran, R., Taagepera

We have determined that the major mitotic phosphoprotein in chromosomes recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa isoform of topoLsomerase II (topo II), the isoform predominat in proHlferating cells. As a prerequisite to making this discovery, it was necesary to develop protocols to protect chromosomal proteins from dephosphorylation during cell extraction and chromosome isoltion procedures During the G2 -* M transition of the cell cycle, the interphase chromatin condenses =10,000-fold into compact mitotic chromosomes containing DNA, histones, and a variety of nonhistone proteins. Extraction of isolated chromosomes with high salt buffers leaves a fraction of nonhistone proteins termed the chromosome scaffold fraction (1). This fraction contains two major proteins, Scl (170 kDa) and Sc2 (135 kDa) (2, 3). Scl was identified as the protein DNA topoisomerase II (topo II) (4, 5), an essential highly conserved protein present in the nuclei of most eukaryotic cells (6). Topo II has been implicated in the regulation of gene structure and function through its ability to alter DNA topology. Functioning as a dimer, topo II catalyzes the passage of one intact DNA helix through a transient double-stranded break in a second DNA helix in an AT?-dependent manner. Topo II appears to be involved in many aspects of nucleic acid metabolism, including initiation of DNA replication (7, 8), DNA repair (9), recombination (10), and transcription (11,12). Additionally, topo II is essential for the restructuring of chromatin in mitosis (13). The condensation ofchromatin into chromosome-like structures in cell-free mitotic extracts is dependent on the activity of topo II (14)(15)(16) condensation during mitosis (17) and for separation of sister chromatids during anaphase (18,19). Topo II may also play a karyoskeletal role in both interphase nuclei and mitotic chromosomes. Some immunofluorescence and immunoelectron microscopy studies of mitotic chromosomes indicate that topo II is concentrated along a dense axial core in the chromosome arms. This localization has been cited as evidence that topo II plays a structural role in the mitotic chromosome. However, this view has recently been challenged by evidence that topo II is evenly distributed throughout the chromosome arms and that its continuous presence is not essential in maintaining the structural integrity of mitotic chromosomes after they have been assembled (20, 21). Recent work has demonstrated the existence of two topo II isoforms in mammalian cells (22)-the 170-kDa isoform (topo Ila) and the 180-kDa isoform (topo II8) (23). These topo II isoforms are products of separate genes (22) and are differentially regulated during cellular proliferation (24).MPM-2 is a monoclonal antibody originally prepared to mitotic HeLa cell extracts (25). MPM-2 recognizes a phosphorylated epitope found predominantly in proteins that are phosphorylated at mitosis (26). Immunofluorescence and immunoelectron microscopy analyses of mitotic cells revealed MPM-2 labeling of so...

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk. Binding of the MPM-2 antibody to active p42mapk in vitro results in a decrease in p42mapk enzymatic activity. The MPM-2 phosphoepitope can be generated in vitro on bacterially expressed p42mapk by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42mapk proteins mutated in their regulatory sites shows that phosphorylated Thr-183 is essential for the binding of the MPM-2 antibody. MPM-2 binding to Thr-183 is affected by the amino acid present in the other regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalanine results in strong binding of the MPM-2 antibody, whereas substitution with glutamic acid substantially diminishes MPM-2 antibody binding. The MPM-2 phosphoepitope antibody recognizes an amino acid domain incorporating the regulatory phosphothreonine on activated p42mapk in eggs during meiosis and in mammalian cultured cells during the G0 to G1 transition.

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