Role of MacMARCKS in Integrin-dependent Macrophage Spreading and Tyrosine Phosphorylation of Paxillin

doi:10.1074/jbc.271.22.12985

Role of MacMARCKS in Integrin-dependent Macrophage Spreading and Tyrosine Phosphorylation of Paxillin*

From the Department of Microbiology and Immunology, University of Tennessee at Memphis, College of Medicine, Memphis, Tennessee 38163

^‡ To whom correspondence should be addressed:
The University of Tennessee, Memphis, Department of Microbiology & Immunology, 858 Madison Ave., Memphis, TN 38163.
Tel.: 901-448-6763; Fax: 901-448-8462; E-mail: jxli{at}utmem2.utmem.edu

Abstract

The cellular function of the MARCKS family of protein kinase C substrates is unknown. In this report, we present evidence that indicates a role for MacMARCKS, a member of the MARCKS family, in the integrin-dependent signal transduction pathways in macrophages. Using a dominant negative mutant of MacMARCKS, we showed that MacMARCKS participates in several integrin-dependent macrophage functions, including the phorbol ester-stimulated macrophage spreading, a process involving multiple integrins. The dominant negative mutant also blocks macrophage spreading on immune complex-coated surfaces, a process again requiring β₂ integrin. More direct evidence of the role of MacMARCKS in the integrin-dependent pathway is the ablation of macrophage binding to complement iC3b-coated sheep erythrocytes by MacMARCKS mutant, suggesting an effect of this mutant on the avidity of complement receptor 3, a member of the β₂ integrin family. To further evaluate the possible mechanism of MacMARCKS function, the integrin-dependent tyrosine phosphorylation of paxillin was examined. Concomitant with the inhibition of macrophage spreading and rosette formation, MacMARCKS mutant also inhibits integrin-dependent tyrosine phosphorylation of paxillin. Furthermore, immunofluorescent microscopy data showed that MacMARCKS and paxillin colocalize in the membrane ruffles at the leading edge of the spreading cells, providing a potential site and opportunity for MacMARCKS to participate in the regulation of integrin-dependent tyrosine phosphorylation of paxillin. Together, these data strongly suggest that MacMARCKS plays a role in integrin-dependent signal transduction pathways in macrophages.

Footnotes

↵* This study was supported by a grant from the Council for Tobacco Research. 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:

SH2 and SH3

Src homology regions 2 and 3, respectively

BSA

bovine serum albumin

ED

effector domain deletion mutant of MacMARCKS

E_iC3b

iC3b-coated sheep erythrocytes

E_IgM

IgM-coated sheep erythrocytes

FM

full-length wild type MacMARCKS

PAGE

polyacrylamide gel electrophoresis

MARCKS

myristoylated alanine-rich protein kinase C substrate

PBS

phosphate-buffered saline

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate.
- Received January 22, 1996.
- Revision received March 21, 1996.