Involvement of p130Cas and p105HEF1, a Novel Cas-like Docking Protein, in a Cytoskeleton-dependent Signaling Pathway Initiated by Ligation of Integrin or Antigen Receptor on Human B Cells

doi:10.1074/jbc.272.7.4230

Involvement of p130^Cas and p105^HEF1, a Novel Cas-like Docking Protein, in a Cytoskeleton-dependent Signaling Pathway Initiated by Ligation of Integrin or Antigen Receptor on Human B Cells*

From the Departments of ^‡ Medicine, Harvard Medical School, Boston, Massachusetts 02115 and
Department of ^¶ Pathology, Harvard Medical School, Boston, Massachusetts 02115 the
** Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 the
^‡‡ Third Department of Internal Medicine, University of Tokyo, Hongo, Tokyo 113, Japan the
^§§ Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201 the
^¶¶ Division of Hematology/Oncology, Deaconess Hospital, Boston, Massachusetts 02115 and the
Divisions of ^§ Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 and
Division of ^∥ Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115

^∥∥ To whom correspondence should be addressed:
Division of Hematologic Malignancies, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115
. Tel.: 617-632-3441; Fax: 617-632-5167.

Abstract

The Crk-associated substrate p130^Cas (Cas) and the recently described human enhancer of filamentation 1 (HEF1) are two proteins with similar structure (64% amino acid homology), which are thought to act as “docking” molecules in intracellular signaling cascades. Both proteins contain an N-terminal Src homology (SH), three domain and a cluster of SH2 binding motifs. Here we show that ligation of either β1 integrin or B cell antigen receptor (BCR) on human tonsillar B cells and B cell lines promoted tyrosine phosphorylation of HEF1. In contrast, Cas tyrosine phosphorylation was observed in certain B cell lines but not in tonsillar B cells, indicating a more general role for HEF1 in B cell signaling. Interestingly, pretreatment of tonsillar B cells with cytochalasin B dramatically reduced both integrin- and BCR-induced HEF1 phosphorylation, suggesting that some component of the BCR-mediated signaling pathway is closely linked with a cytoskeletal reorganization. Both HEF1 and Cas were found to complex with the related adhesion focal tyrosine kinase (RAFTK), and when tyrosine phosphorylated, with the adapter molecule CrkL. In addition, the two molecules were detected in p53/56^Lyn immunoprecipitates, and Lyn kinase was found to specifically bind the C-terminal proline-rich sequence of Cas in an in vitro binding assay. These associations implicate HEF1 and Cas as important components in a cytoskeleton-linked signaling pathway initiated by ligation of β1 integrin or BCR on human B cells.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants CA55207 and CA66996 and American Cancer Society Grant DHP-145 (to A. S. F.), a Fellowship from the Lymphoma Foundation of America (to S. N. M.), National Institutes of Health Grants CA60821 (to R. S.), CA36167 (to J. D. G.), and HL55445 (to H. A.), National Institutes of Health Training Grant CA09035 (to S. F. L), and American Cancer Society Grant CB-74749 and National Institutes of Health Grant R29-CA63366 (to E. A. G.). 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:

FAK

p125^FAK

Cas

p130^Cas

HEF1

human enhancer of filamentation 1

SH

src homology

BCR

B cell antigen receptor

RAFTK

related adhesion focal tyrosine kinase

Cbl

p120^c-CBL

RAM

rabbit anti-mouse Ig

CB

cytochalasin B

anti-P-tyr

anti-phosphotyrosine

IVK

in vitro kinase assay

PAGE

polyacrylamide gel electrophoresis.
↵² During the time of revision of this manuscript, Minegishi, M., Tachibana, K., Sato, T., Iwata, S., Nojima, Y., and Morimoto, C. (1996) J. Exp. Med. 184, 1365, reported the cloning of Cas-L, which is identical to HEF1, and is tyrosine phosphorylated following β1 integrin ligation in T cells.
↵³ S. N. Manie, A. Astier, and A. S. Freedman, unpublished data.
↵⁴ A. Astier, S. N. Manie, S. F. Law, T. Canty, N. Haghayeghi, B. J. Druker, R. Salgia, E. A. Golemis, and A. S. Freedman, submitted for publication.
- Received August 7, 1996.
- Revision received November 11, 1996.