Characterization of clathrin and Syk interaction upon Shiga toxin binding.
Shiga toxin (Stx) is a bacterial toxin that binds to its receptor Gb3 at the plasma membrane. It is taken up by endocytosis and transported retrogradely via the Golgi apparatus to the endoplasmic reticulum. The toxin is then translocated to the cytosol where it exerts its toxic effect. We have previously shown that phosphorylation of clathrin heavy chain (CHC) is an early event following Stx binding to HeLa cells, and that this requires the activity of the tyrosine kinase Syk. Here, we have investigated this event in more detail in the B lymphoid cell line Ramos, which expresses high endogenous levels of both Syk and Gb3. We report that efficient endocytosis of Stx in Ramos cells requires Syk activity and that Syk is recruited to the uptake site of Stx. Furthermore, in response to Stx treatment, CHC and Syk were rapidly phosphorylated in a Src family kinase dependent manner at Y1477 and Y352, respectively. We show that these phosphorylated residues act as binding sites for the direct interaction between Syk and CHC. Interestingly, Syk-CHC complex formation could be induced by both Stx and B cell receptor stimulation.
Wälchli
Sébastien
S
Department of Biochemistry, Institute for Cancer Research, Faculty Division: The Norwegian Radium Hospital, Montebello, Oslo, Norway. sebastien.walchli@rr-research.no
Aasheim
Hans-Christian
HC
Skånland
Sigrid S
SS
Spilsberg
Bjørn
B
Torgersen
Maria L
ML
Rosendal
Ken R
KR
Sandvig
Kirsten
K
D001665:Binding Sites; D002966:Clathrin / Q000378:metabolism*; D033922:Clathrin Heavy Chains / Q000378:metabolism; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D022621:Shiga Toxin / Q000378:metabolism*; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D002966:Clathrin; D047908:Intracellular Signaling Peptides and Proteins; D033922:Clathrin Heavy Chains; D014443:Tyrosine; D022621:Shiga Toxin; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D019061:src-Family Kinases