Centrosomal targeting of Syk kinase is controlled by its catalytic activity and depends on microtubules and the dynein motor

Abstract

The nonreceptor Syk kinase is detected in epithelial cells, where it acts as a tumor suppressor, in addition to its well-established role in immunoreceptor-based signal transduction in hematopoietic cells. Thus, several carcinomas and melanomas have subnormal concentrations of Syk. Although Syk is mainly localized at the plasma membrane, it is also present in centrosomes, where it is involved in the control of cell division. The mechanisms responsible for its centrosomal localization and action are unknown. We used wild-type and mutant fluorescent Syk fusion proteins in live-cell imaging (fluorescence recovery after photobleaching, total internal reflection fluorescence, and photoactivation) combined with mathematical modeling to demonstrate that Syk is actively transported to the centrosomes via the microtubules and that this transport depends on the dynein/dynactin molecular motor. Syk can only target the centrosomes if its kinase activity is intact and it is catalytically active at the centrosomes. We showed that the autophosphorylated Y130 Syk residue helps to uncouple Syk from the plasma membrane and to promote its translocation to the centrosome, suggesting that the subcellular location of Syk depends on its autophosphorylation on specific tyrosine residues. We have thus established the details of how Syk is trafficked intracellularly and found evidence that its targeting to the centrosomes is controlled by autophosphorylation.

Nanomechanical Property Maps of Breast Cancer Cells As Determined by Multiharmonic Atomic Force Microscopy Reveal Syk-Dependent Changes in Microtubule Stability Mediated by MAP1B

“… 44 , 52 , 53 The movement of Syk to the centrosome is dependent on both the catalytic activity of Syk and its movement along microtubules mediated by the dynein–dynactin complex. 54 A phosphoproteomic analysis of Syk-dependent substrates identified several centrosomal and microtubule-associated proteins. 24 , 44 The substrate with the most identified sites of phosphorylation was MAP1B, a microtubule-associated protein found most prominently in the nervous system.…”

The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex

“…The FLAG-tagged Syk [20], the pEGFP-Syk [71], the mutant K402R DsRed-Syk [72], and E-cadherin-GFP (Addgene, Watertown, MA, USA) [73] constructs were previously described. The pEGFP-Syk-rescue mutant construct was obtained by generating a silencing mutation (CATCATCAGTCAGAA to CATCATTTCTCAGAA) in the SYK sequence using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and confirmed by sequencing.…”

Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1

“…On the other hand, SHP-1 regulated the activity of the Syk kinase (Figure 5), which was reported to interact with γ-tubulin [6]. Moreover, in breast cancer cells, Syk localized to centrosomes [46,47]. We, therefore, investigated whether, in BMMCs, Syk forms complexes with SHP-1 and localizes to centrosomes.…”

“…Androgen and Src signaling modulated the microtubule nucleation during interphase by promoting the centrosomal localization of γ-tubulin [16] via the activation of the MAPK/Erk signaling pathway [17]. It has also been shown that Syk is catalytically active at the centrosome [46,47]. In the early stages of BMMCs activation, when microtubule formation is stimulated, tyrosine-phosphorylated proteins concentrate in the centrosomal region.…”