Syk Tyrosine Kinase Is Required for Immunoreceptor Tyrosine Activation Motif-dependent Actin Assembly

doi:10.1074/jbc.271.28.16597

Syk Tyrosine Kinase Is Required for Immunoreceptor Tyrosine Activation Motif-dependent Actin Assembly*

From the Department of ^‡ Medicine, Pulmonary Division, College of Physicians and Surgeons, Columbia University, New York, New York 10032, the Department of
^§ Cardiovascular Molecular Biology, Lederle Laboratories, Pearl River, New York, New York 10965, and Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06510

^¶ To whom correspondence should be addressed:
Columbia University, Dept. of Medicine, 630 West 168th St., New York, NY 10032.
Tel.: 212-305-1586; Fax: 212-305-1146; E-mail: greenberg{at}cuccfa.ccc.columbia.edu

Abstract

Clustering of several multisubunit receptors on hematopoetic cells results in a signaling cascade involving the phosphorylation of immunoreceptor tyrosine activation motifs, or “ITAMs,” and actin polymerization. Recent experiments indicate that direct clustering of the ITAM-binding protein, p72^syk (Syk), is capable of transmitting a phagocytic signal in COS cells (Greenberg, S., Chang, P., Wang, D., Xavier, R., and Seed, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1103-1107). However, the possibility of redundant signaling pathways makes it difficult to test the requirement for Syk in ITAM-dependent actin polymerization in hematopoetic cells. We developed a model system to study ITAM-dependent actin assembly. DT40 lymphocytes were transfected with fusion proteins encoding the transmembrane and cytosolic domains of the ITAM-containing γ subunit of Fc receptors. Clustering the γ-containing fusion proteins with IgG-coated erythrocytes triggered submembranous actin assembly. This response depended on an intact ITAM, was absent in cell lines that had been engineered to lack Syk, and was augmented in cell lines that stably overexpressed Syk. These experiments demonstrate an absolute requirement for Syk tyrosine kinase in ITAM-dependent actin assembly in transfected lymphocytes.

Footnotes

↵* This work was supported in part by Grants HL02641 and HL54164 from the National Institutes of Health, a Research Grant from the American Cancer Society, and a grant-in-aid from the American Heart Association, New York City affiliate. 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:

Fc_γ receptor

receptor for the Fc portion of IgG

ITAM

immunoreceptor tyrosine activation motif

mAb

monoclonal antibody

RBCs

red blood cells

IgG-RBCs

sheep erythrocytes opsonized with anti-sheep erythrocyte IgG

Syk⁺⁺

DT40 cell lines that overexpress Syk

Syk⁻

DT40 cell lines that lack expression of Syk

16:γ

fusion proteins containing CD16 ectodomains and γ subunit transmembrane and cytosolic domains

16:γ_{Y → F}

a 16:γ-based construct bearing a Tyr → Phe mutation in tyrosine 76

WT

wild-type DT40 cells.
- Received March 5, 1996.
- Revision received April 22, 1996.