M-CSF Regulates the Cytoskeleton via Recruitment of a Multimeric Signaling Complex to c-Fms Tyr-559/697/721

Roberta Faccio,Jean Chappel,Alberta Zallone,Graziana Colaianni,Sunao Takeshita,Steven L Teitelbaum,F Patrick Ross

doi:10.1074/jbc.m610937200

Abstract

M-CSF is known to induce cytoskeletal reorganization in macrophages and osteoclasts by activation of phosphatidylinositol 3-kinase (PI3K) and c-Src, but the detailed mechanisms remain unclear. We find, unexpectedly, that tyrosine (Tyr) to phenylalanine (Phe) mutation of Tyr-721, the PI3K binding site in the M-CSF receptor c-Fms, fails to suppress cytoskeletal remodeling or actin ring formation. In contrast, mutation of c-Fms Tyr-559 to Phe blocks M-CSF-induced cytoskeletal reorganization by inhibiting formation of a Src Family Kinase SFK.c-Cbl.PI3K complex and the downstream activation of Vav3 and Rac, two key mediators of actin remodeling. Using an add-back approach in which specific Tyr residues are reinserted into c-Fms inactivated by the absence of all seven functionally important Tyr residues, we find that Tyr-559 is necessary but not sufficient to transduce M-CSF-dependent cytoskeletal reorganization. Furthermore, this same add-back approach identifies important roles for Tyr-697 and Tyr-721 in collaborating with Tyr-559 to recruit a multimeric signaling complex that can transduce signals from c-Fms to the actin cytoskeleton.

Highlights

In migrating macrophages and OCs, podosomes are confined to the periphery of the cell, where their reorganization results in lamellipodia formation, a process dependent on de novo actin assembly by the Arp2/3-Ena1⁄7Wiskott-Aldrich syndrome protein complex [8]
M-CSF-dependent Cytoskeletal Organization in Osteoclasts Does Not Require Direct Recruitment of phosphatidylinositol 3-kinase (PI3K) to c-Fms Tyr-721— PI3K mediates M-CSF-dependent cytoskeletal organization [31, 32], the c-Fms tyrosine residue/s that regulate the cytoskeletal reorganization in response to M-CSF in OCs have not been identified
We generated OCs from primary Bone marrow-derived macrophages (BMMs) transduced with the WT or the Phe721-mutated chimera in the presence of RANKL and M-CSF and treated these cells with Epo to examine subsequent cytoskeletal reorganization dependent on the chimeric EpoR/ c-Fms receptor

Summary

Introduction

In migrating macrophages and OCs, podosomes are confined to the periphery of the cell, where their reorganization results in lamellipodia formation, a process dependent on de novo actin assembly by the Arp2/3-Ena1⁄7Wiskott-Aldrich syndrome protein complex [8]. The fact that podosome formation, macrophage chemotaxis, and osteoclast movement on bone are impaired by mutation of the Wiskott-Aldrich syndrome protein [11], inhibition of Rac activity [12], or small interfering RNA suppression of the actinrelated protein 2/3 complex [13, 14] attests to the importance of actin assembly in macrophages and OC function. Several Src homology 2 domain-containing molecules are recruited to the phospho-Tyr residues upon M-CSF binding and initiate different signaling cascades that lead to cell proliferation, differentiation, or cytoskeletal reorganization [17]. The detailed signaling cascade activated by M-CSF is not yet fully understood, PI3K and SFKs are recruited to c-Fms and regulate migration of macrophages and OCs. Migration of PI3K␥- or PI3K␣-null macrophages is impaired in response to the cytokine [21, 22]. The M1 myeloid cell line, which cannot differentiate into mature osteoclasts, displays defective M-CSF-dependent differentiation into more mature macrophages when overexpressing a specific c-Fms tyrosine mutant (Phe-559) that lacks the ability to recruit Src family members, highlighting the importance of c-Fms Tyr-559 and Src family-dependent signaling [27]

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Conclusion