Biomedical subjects

L L Whitaker

Publications and source records attributed to L L Whitaker.

5 recordsLinked to original sources

PubMed1998

Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase.

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, S., Schwartz, M. & Wang, J. (1996) Proc. Natl. Acad. Sci. USA 93, 15174-15179]. Here we show that the C terminus of c-Abl does indeed contain a functional nuclear export signal (NES) with the characteristic leucine-rich motif. The c-Abl NES can functionally complement an NES-defective HIV Rev protein (RevDelta3NI) and can mediate the nuclear export of glutathione-S-transferase. The c-Abl NES function is sensitive to the nuclear export inhibitor leptomycin B. Mutation of a single leucine (L1064A) in the c-Abl NES abrogates export function. The NES-mutated c-Abl, termed c-Abl NES(-), is localized exclusively to the nucleus. Treatment of cells with leptomycin B also leads to the nuclear accumulation of wild-type c-Abl protein. The c-Abl NES(-) is not lost from the nucleus when detached fibroblasts are replated onto fibronectin matrix. Taken together, these results demonstrate that c-Abl shuttles continuously between the nucleus and the cytoplasm and that the rate of nuclear import and export can be modulated by the adherence status of fibroblastic cells.

3T3 Cells↗

PubMed1998

Growth suppression by an E2F-binding-defective retinoblastoma protein (RB): contribution from the RB C pocket.

Growth suppression by the retinoblastoma protein (RB) is dependent on its ability to form complexes with transcription regulators. At least three distinct protein-binding activities have been identified in RB: the large A/B pocket binds E2F, the A/B pocket binds the LXCXE peptide motif, and the C pocket binds the nuclear c-Abl tyrosine kinase. Substitution of Trp for Arg 661 in the B region of RB (mutant 661) inactivates both E2F and LXCXE binding. The tumor suppression function of mutant 661 is not abolished, because this allele predisposes its carriers to retinoblastoma development with a low penetrance. In cell-based assays, 661 is shown to inhibit G1/S progression. This low-penetrance mutant also induces terminal growth arrest with reduced but detectable activity. We have constructed mutations that disrupt C pocket activity. When overproduced, the RB C-terminal fragment did not induce terminal growth arrest but could inhibit G1/S progression, and this activity was abolished by the C-pocket mutations. In full-length RB, the C-pocket mutations reduced but did not abolish RB function. Interestingly, combination of the C-pocket and 661 mutations completely abolished RB's ability to cause an increase in the percentage of cells in G1 and to induce terminal growth arrest. These results suggest that the A/B or C region can induce a prolongation of G1 through mechanisms that are independent of each other. In contrast, long-term growth arrest requires combined activities from both regions of RB. In addition, E2F and LXCXE binding are not the only mechanisms through which RB inhibits cell growth. The C pocket also contributes to RB-mediated growth suppression.

Binding Sites↗

PubMed1997

Induction of apoptosis in Mv1Lu cells by expression of competitive RB1 mutants.

The Retinoblastoma tumor suppressor gene (RB1) plays a role in a variety of human cancers. Experimental analyses have indicated that the protein product of the RB1 gene (pRb) plays a role in cell cycle regulation. pRb function is dependent on its ability to bind to cellular factors, which is facilitated by multiple protein binding domains within pRb. Mutations within these domains which eliminate the ability of pRb to bind its targets result in loss of function. Although loss of pRb function may lead to uncontrolled cellular proliferation, tumorigenesis is not the only response to pRb inactivation. Examination of various tissues from RB1-nullizygous mouse embryos showed problems in differentiation and induction of apoptosis, suggesting that the ultimate response to the loss of pRb is influenced by cellular context. It has previously been demonstrated in RB1-negative Saos-2 cells that co-expression of the C-terminal domain of pRb with wildtype pRb resulted in escape from G1 arrest and continued cell cycling. In this paper we demonstrate that in RB1-positive Mv1Lu cells, expression of the A/B pocket region or the N-terminus (when combined with a nuclear localization signal), but not the C-terminal domain, is able to competitively disrupt the function of the endogenous pRb, and that the result of this disruption is apoptosis.

Animals↗

PubMed1997

Ataxia telangiectasia mutant protein activates c-Abl tyrosine kinase in response to ionizing radiation.

Ataxia telangiectasia (AT) is a rare human autosomal recessive disorder with pleiotropic phenotypes, including neuronal degeneration, immune dysfunction, premature ageing and increased cancer risk. The gene mutated in AT, ATM, encodes a putative lipid or protein kinase. Most of the human AT patient phenotypes are recapitulated in Atm-deficient mice. Cells derived from Atm-/- mice, like those from AT patients, exhibit abnormal response to ionizing radiation. One of the known responses to ionizing radiation is the activation of a nuclear tyrosine kinase encoded by the c-abl proto-oncogene. Ionizing radiation does not activate c-Abl in cells from AT patients or in thymocytes or fibroblasts from the Atm-deficient mice. Ectopic expression of a functional ATM kinase domain corrects this defect, as it phosphorylates the c-Abl tyrosine kinase in vitro at Ser 465, leading to the activation of c-Abl. A mutant c-Abl with Ser 465 changed to Ala 465 is not activated by ionizing radiation or ATM kinase in vivo. These findings identify the c-Abl tyrosine kinase as a downstream target of phosphorylation and activation by the ATM kinase in the cellular response to ionizing radiation.

3T3 Cells↗

PubMed1993

A method to isolate DNA from small archival tissue samples for p53 gene analysis.

The tumor suppressor gene p53 is involved in predisposition to a variety of human cancers, including those from Li-Fraumeni cancer family syndrome patients. Studies of inheritance of p53 germline mutations require confirmation of the mutation in the tumors from family members. These studies, as well as other retrospective studies of tumor specific mutations, are often hampered by a lack of available fresh or frozen tumor tissue samples for DNA extraction to confirm the suspected p53 mutation. Here we describe a simple technique for DNA isolation that permits mutational analysis of p53 from minimal amounts of paraffin-embedded archival tissue samples.

Base Sequence↗