Thrombin-induced tyrosine phosphorylation of HS1 in human platelets is sequentially catalyzed by Syk and Lyn tyrosine kinases and associated with the cellular migration of the protein.
Thrombin stimulation of platelets triggers Tyr phosphorylation of several signaling proteins, most of which remain unidentified. In this study, we demonstrate for the first time that hematopoietic lineage cell-specific protein 1 (HS1) undergoes a transient Tyr phosphorylation in human platelets stimulated with thrombin. The protein is synergistically phosphorylated by Syk and Lyn tyrosine kinases according to a sequential phosphorylation mechanism. By means of specific inhibitors (PP2, SU6656, and piceatannol) and phosphopeptide-specific antibodies, as well as by coimmunoprecipitation and binding competition experiments, we show that Syk acts as the primary kinase that phosphorylates HS1 at Tyr397 and that Syk phosphorylation is required for HS1 interaction with the Lyn SH2 domain. Upon docking to Syk-phosphorylated HS1, Lyn catalyzes the secondary phosphorylation of the protein at Tyr222. Once the secondary Tyr phosphorylation of HS1 is accomplished the protein dissociates from Lyn and undergoes a dephosphorylation process. HS1 Tyr phosphorylation does not occur when thrombin-induced actin assembly is inhibited by cytochalasin D even under conditions in which Syk and Lyn are still active. Immunofluorescence microscopic analysis shows that the agonist promotes HS1 migration to the plasma membrane and that the inhibition of Lyn-mediated secondary phosphorylation of HS1 abrogates the subcellular translocation of the protein. All together these results indicate that HS1 Tyr phosphorylation catalyzed by Syk and Lyn plays a crucial role in the translocation of the protein to the membrane and is involved in the cytoskeleton rearrangement triggered by thrombin in human platelets.
Brunati
Anna Maria
AM
Department of Biochemistry, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.
Deana
Renzo
R
Folda
Alessandra
A
Massimino
Maria Lina
ML
Marin
Oriano
O
Ledro
Silvia
S
Pinna
Lorenzo A
LA
Donella-Deana
Arianna
A
D017209:Apoptosis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D015638:Cytochalasin D / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003720:Densitometry; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013347:Subcellular Fractions; D000072377:Syk Kinase; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry*; D019061:src-Family Kinases / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004792:Enzyme Precursors; C031908:H-NS protein, bacteria; D047908:Intracellular Signaling Peptides and Proteins; D015638:Cytochalasin D; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D013917:Thrombin