The Redistribution of Cortactin into Cell-Matrix Contact Sites in Human Carcinoma Cells with 11q13 Amplification Is Associated with Both Overexpression and Post-translational Modification

doi:10.1074/jbc.272.11.7374

The Redistribution of Cortactin into Cell-Matrix Contact Sites in Human Carcinoma Cells with 11q13 Amplification Is Associated with Both Overexpression and Post-translational Modification*

From the Department of Pathology, Leiden University, Leiden, The Netherlands

^‡ To whom correspondence should be addressed:
Dept. of Pathology, University of Leiden, L1Q, P. O. Box 9600, 2300 RC Leiden, The Netherlands.
Tel.: 31-715-266629; Fax: 31-715-248158; E-mail:

Abstract

The EMS1 gene, located at the chromosome 11q13 region, is the human homologue of p80/p85 cortactin, a chicken pp60^src tyrosine kinase substrate. In cells derived from breast carcinomas and squamous carcinomas of the head and neck, DNA amplification of this region results in overexpression of cortactin. Overexpression is accompanied by a partial redistribution of cortactin from the cytoplasm into cell-matrix contact sites. To investigate whether overexpression only is sufficient for this redistribution, we performed biochemical analysis of human cortactin derived from carcinoma cell lines with either normal levels (UMSCC8) or with excessive levels of cortactin due to chromosome 11q13 amplification (UMSCC2). Pulse-chase experiments performed with UMSCC2 cells revealed that p85 originated from p80 by post-translational modifications. However, the conversion of p80 into p85 was hardly observed in UMSCC8 cells, indicating a different processing of the two isoforms in cells with a normal expression level of cortactin. Western blot analysis showed that treatment of UMSCC2 cells with cycloheximide, serum, epidermal growth factor, or vanadate resulted in the disappearance of the p80 form and conversion into p85. Conversion of p80 into p85 was accompanied by a redistribution of cortactin from cytoplasm to cell-matrix contact sites. In UMSCC8 cells, these treatments had no effect on the p80/p85 ratio, and cortactin remained in the cytoplasm. Conversion into p85 therefore is correlated with a relocalization of cortactin to the cell periphery. In addition, p85 from epidermal growth factor- or vanadate-treated UMSCC2 cells showed a significant enhancement in phosphorylation compared with p85 in UMSCC8 cells. Our findings demonstrate that in carcinoma cells with 11q13 amplification not only overexpression but also post-translational modifications of cortactin coincides with the redistribution from the cytoplasm into cell-matrix contact sites.

Footnotes

↵* This work was supported by Grant RUL93-550 from The Dutch Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EGF

epidermal growth factor

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

PVDF

polyvinylidene difluoride.
↵² Schuuring, H. van Damme, H. Brok, and E. Schuuring-Scholtes, unpublished observations.
↵³ B. Verbeek and G. Rijksen, unpublished observations.
↵⁴ H. van Damme, V. van Buuren, E. Schuuring-Scholtes, and E. Schuuring, manuscript in preparation.
- Received July 17, 1996.
- Revision received November 12, 1996.