Raft-mediated Src homology 2 domain-containing proteintyrosine phosphatase 2 (SHP-2) regulation in microglia.
Janus kinase-signal transducer and activator of transcription (JAK-STAT) signals play important roles in cell proliferation, apoptosis, and inflammation, and they recently have been considered as therapeutic targets for suppressing oncogenesis and inflammatory process. Phosphatases including Src homology 2 domain-containing protein-tyrosine phosphatases (SHPs), are well known as negative regulators of the JAK-STAT pathway, but their precise mechanisms are largely unknown. Based on our previous finding that in cultured rat brain microglia, gangliosides induce rapid and transient activation of the JAK-STAT pathway, we hypothesized that raft-mediated SHP-2 activation is involved in transient activation of JAK-STAT signaling by gangliosides. We first used Western blot analysis to confirm that gangliosides rapidly induce the phosphorylation of SHP-2. This was inhibited by pretreatment with the lipid raft disrupter filipin and was restored following filipin removal. Immunostaining using antibodies directed against p-SHP-2 and flotillin-1 revealed ganglioside-induced clustering and polarization of p-SHP-2 in membrane rafts. Raft-associated regulation of SHP-2 was further demonstrated in fractionation experiments using detergent and detergent-free sucrose gradient ultracentrifugation. Rapid SHP-2 recruitment to detergent-insoluble raft fractions by gangliosides was inhibited by filipin, further indicating the involvement of rafts. We also confirmed by immunoprecipitation that SHP-2 rapidly binds in a raft-dependent manner to JAK2 in response to gangliosides. Our study therefore showed that transient activation of the JAK-STAT pathway by gangliosides is accomplished by SHP-2 in a raft-dependent manner in brain microglia.
Kim
Hee Young
HY
Department of Pharmacology and Chronic Inflammatory Disease Research Center, Ajou University School of Medicine, Suwon 442-721, Korea.
Park
Soo Jung
SJ
Joe
Eun-hye
EH
Jou
Ilo
I
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005372:Filipin / Q000494:pharmacology; D005732:Gangliosides / Q000378:metabolism; D005786:Gene Expression Regulation; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D053614:Janus Kinase 2; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000201:enzymology*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D018909:src Homology Domains
D005732:Gangliosides; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D050796:STAT3 Transcription Factor; C106354:flotillins; D005372:Filipin; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; C507927:Jak2 protein, rat; D053614:Janus Kinase 2; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C516727:Ptpn11 protein, rat; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases