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Multiple phosphorylation of -synuclein by protein tyrosine kinase Syk prevents eosin-induced aggregation ¹

Dipartimento di Chimica Biologica and Centro di Studio delle Biomembrane del C.N.R., University of Padova, Padova, Italy

²Correspondence: Dipartimento di Chimica Biologica, Viale G, Colombo, 3, 35121 Padova, Italy. E-mail: pinna{at}civ.bio.unipd.it

SPECIFIC AIMS

This work was performed to investigate the possibility that -synuclein might undergo stoichiometric tyrosine phosphorylation and to assess whether such a phosphorylation affects its tendency to multimerize, which is believed to underlie the neurodegenerative potential of this protein. Given the presence of three tyrosyl residues in the carboxyl-terminal segment of -synuclein that display the consensus sequence recognized by the protein tyrosine kinase p72^syk, we wanted to see how phosphorylation by Syk compares with that catalyzed by other kinases both in terms of catalytic efficiency and functional consequences. Moreover, we outlined physical interactions between Syk and -synuclein in transfected cells.

PRINCIPAL FINDINGS

1. In vitro -synuclein is readily phosphorylated by Syk to a stoichiometry approaching 2 mol phosphate/mol protein and a low K_m value (50 nM)
In contrast, phosphorylation by two tyrosine kinases of the Src family—c-Fgr and Lyn—is largely understoichiometric (<0.3 mol P/mol protein) and occurs with less favorable kinetics (K_m=0.5 µM). Phosphorylation by two acidophilic Ser/Thr kinases, CK2 and G-CK, is also negligible compared with Syk, whereas no phosphorylation could be detected with protein kinases CK1 and Csk under our conditions.

2. Y125, Y133, and Y136 are affected by Syk
Mutational analysis of tyrosyl residues in the carboxyl-terminal segment of -synuclein (Y125, Y133, and Y136) reveals that these three residues, but not the tyrosine present in the amino-terminal moiety (Y39), are affected by Syk, albeit with variable efficiencies (Fig. 1 A). By contrast, only Y125 is phosphorylated by Lyn (Fig. 1B ) and c-Fgr (not shown), in agreement with the finding by others that Y125 alone is phosphorylated by the Src-related tyrosine kinases Src and Fyn.

View larger version (35K): [in this window] [in a new window]   Figure 1. Phosphorylation of wild-type -synuclein and its mutants by Syk and Lyn tyrosine kinases. 0.25 µM wild-type -synuclein and -synuclein mutants were phosphorylated for 30 min by either 10 nM Syk (A) or 50 nM Lyn (B). Reported values represent the means of three separate experiments, with SE indicated by vertical bars.

3. Self-oligomerization of -synuclein
As judged from the eosin-induced cross-linking method, self-oligomerization is suppressed if -synuclein is previously phosphorylated by Syk but not if it is phosphorylated by Src-related tyrosine kinases Lyn and c-Fgr (Fig. 2 ). This would indicate that multiphosphorylation at its carboxyl-terminal tyrosines causes a conformational alteration that renders the central hydrophobic region responsible for eosin-assisted multimerization less exposed. This point of view is corroborated by the observation that -syn assembly into filaments is counteracted by previous phosphorylation by Syk, as judged by centrifugation experiments.

View larger version (35K): [in this window] [in a new window]   Figure 2. Tyr-phosphorylation catalyzed by Syk prevents -synuclein multimerization. A) 50 pmol of either wild-type -synuclein (lanes 1, 2) or mutant -syn (YYY125,133,136FFF) (lanes 3, 4) was incubated in the absence (lanes 1, 3) or presence (lanes 2, 4) of eosin (molar ration of 1:20) at pH 6.5 for 40 min at 37°C. Reactions were then incubated for l h at 37°C with 0.3 mM EEDQ stocked in DMSO. After incubation, samples were subjected to 15% SDS/PAGE and immunostained with anti--synuclein antibody. (B, C) 50 pmol of -synuclein was phosphorylated by Syk or Lyn in the presence of [32P]ATP. The stoichiometry of phosphorylation was 0.3 (lanes 1, 2), 1.5 (lanes 3, 4), and 0.3 (lanes 5, 6) mol P/mol -synuclein. After phosphorylation, samples were incubated in the absence (lanes 1, 3, 5) or presence (lanes 2, 4, 6) of eosin and EEDQ, subjected to SDS/PAGE, and transferred to nitrocellulose membrane. The membrane was either immunostained with anti--synuclein antibody (B) or analyzed by autoradiography (C). The figure is representative of five separate experiments.

4. Syk and -synuclein colocalize in the same subcellular compartments
Syk and -synuclein colocalize in the same subcellular compartments, as judged from experiments where neuroblastoma and CHO cells stably transfected with the plasmid pDSsyn encoding a fusion protein between -synuclein and Red Fluorescent Protein were further transiently transfected with the plasmid pEGFPSyk encoding a fusion protein between Syk and Green Fluorescent Protein. Double labeling with antibodies to Syk and -syn on brain sections is also consistent with colocalization of the two proteins. Precise colocalization was confirmed in both experiments by generation of the expected yellow color when the two images were superimposed.

5. Intracellular physical association between Syk and -synuclein
The intracellular physical association between Syk and -synuclein was assessed by the mammalian two-hybrid assay, showing that transcription of the reporter gene CAT occurred only when plasmids pMsyn and pV16Syk, together with the reported plasmid pG5CAT, were both present in the cell.

6. In situ Tyr phosphorylation of -synuclein
Associated with the particulate fraction of CHO cells, -synuclein was dramatically Tyr-phosphorylated if Syk was cotransfected with it, as assessed by Western blot experiments with anti-phosphotyrosine antibody of the different subcellular fractions.

CONCLUSIONS AND SIGNIFICANCE

The data summarized above provide evidence that -synuclein is an outstanding substrate for p72^syk protein tyrosine kinase. At variance with other protein kinases, either Tyr or Ser/Thr specific, whose phosphorylation of -synuclein remains largely understoichiometric, Syk readily phosphorylates -synuclein to a stoichiometry of nearly 2 mol P/mol protein and affects all three tyrosyl residues located in the carboxyl-terminal segment (Y125, Y133, Y136), albeit with different efficiencies. Functional relationships between Syk and -synuclein are also highlighted by the finding that -synuclein colocalizes with Syk both in different kinds of transfected cells and brain sections and that physical interaction between the two proteins could be detected using the mammalian two-hybrid assay. The data are consistent with the scenario schematically outlined in Fig. 3 , whereby Syk or a tyrosine kinase with similar specificity, once activated in the cell, acts as the natural phosphorylating agent of -synuclein. As a result, the multimerization of -synuclein will be prevented, by analogy with the in vitro experiments where aggregation is suppressed by Syk-mediated phosphorylation but not by phosphorylation catalyzed by two other protein tyrosine kinases, Lyn and c-Fgr. These observations disclose new perspectives about the biochemical events affecting the genesis of -synucleinophaties. Our data suggest that the tyrosine kinase Syk, which is abundant in the brain, may also act as an anti-neurodegenerative agent by counteracting the formation of -synuclein aggregates causative of brain lesions. It will be interesting to see whether the etiology of neurodegenerative disorders, with special reference to -synucleinophaties, could be related to impaired activity of Syk or to increased activity of the phosphatase(s) reversing the phosphorylation of -synuclein (see Fig. 3 ). The identification of these latter will represent a crucial challenge in future research on the functional significance of -synuclein phosphorylation.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized role of protein tyrosine kinase Syk as an anti-neurodegenerative agent. Left: a view of the events leading to Syk activation. These are likely to imply the previous activation of Src kinases and/or the phosphorylation of ITAM motifs by analogy to Syk activation in hematopoietic cells. Once activated, Syk interacts with and phosphorylates -synuclein at three tyrosyl residues located in its carboxyl-terminal segment; by so doing, it converts -synuclein into a phospho-form refractory to polymerization. Thus, the formation of aggregates believed to cause -synucleinophaties would be prevented. Phosphorylation of -synuclein by Syk could be potentiated by previous phosphorylation of Ser129 by CK2 or G protein-coupled receptor protein kinases while being reversed by presently unknown tyrosine protein phosphatase(s).

Attention should also focus on circumstances that could lead to decreased Syk activity in neuronal cells. Besides possible structural alterations of Syk, these include impairments in the upstream events leading to Syk activation, notably association with phosphorylated ITAM modules and/or direct phosphorylation by Src family kinases (see Fig. 3 ). The finding by others that PP2, a specific inhibitor of Src kinases, reduces the phosphorylation of -synuclein in HEK293T cells is also quite compatible with our scheme, where activation of Syk is a prerequisite in order to achieve -synuclein phosphorylation. It should be considered that the phosphorylation of Ser129, detected in vivo and catalyzed in vitro by CK2 and GRKs, could have a synergistic effect on the phosphorylation of the tyrosyl residues nearby (especially Y133) inasmuch as upstream phosphoserine has been shown to operate as a positive determinant in Syk-mediated phosphorylation. Alternatively, the phosphorylation of Ser129 may also impinge on the efficiency of -synuclein phosphotyrosine dephosphorylation by protein phosphatases.

In conjunction with the cross-linking of -synuclein via oxidation of its carboxyl-terminal tyrosines to form o,o'-dityrosine as reported by others, the data above highlight the crucial role of the carboxyl-terminal segment as a checkpoint where different signaling pathways can integrate to decide the metabolic fate of -synuclein.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0517fje; to cite this article, use FASEB J. (December 14, 2001) 10.1096/fj.01-0517fje

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