Src-mediated tyrosine phosphorylation of PRC1 and kinastrin/SKAP on the mitotic spindle

Src-family tyrosine kinases (SFKs) play important roles in a number of signal transduction events during mitosis, such as spindle formation. A relationship has been reported between SFKs and the mitotic spindle; however, the underlying mechanisms remain unclear. We herein demonstrated that SFKs accumulated in the centrosome region at the onset of mitosis. Centrosomal Fyn increased in the G2 phase in a microtubule polymerization-dependent manner. A mass spectrometry analysis using mitotic spindle preparations was performed to identify tyrosine-phosphorylated substrates. Protein regulator of cytokinesis 1 (PRC1) and kinastrin/small kinetochore-associated protein (kinastrin/SKAP) were identified as SFK substrates. SFKs mainly phosphorylated PRC1 at Tyr-464 and kinastrin at Tyr-87. Although wild-type PRC1 is associated with microtubules, phosphomimetic PRC1 impaired the ability to bind microtubules. Phosphomimetic kinastrin at Tyr-87 also impaired binding with microtubules. Collectively, these results suggest that tyrosine phosphorylation of PRC1 and kinastrin plays a role in their delocalization from microtubules during mitosis.

Purification of a Src family tyrosine protein kinase from bovine retinas

Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62 kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: ω-aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and α-casein-agarose affinity chromatography. The purified protein was subjected to N-terminal amino acid sequencing and the sequence Gly-Ile-Ile-Lys-Ser-Glu-Glu was obtained, which displayed homology with the first seven residues of the Src family tyrosine kinase c-Yes from Bos taurus (Gly-Cys-Ile-Lys-Ser-Lys-Glu). Although the cytosolic fraction from dark-adapted retinas contained tyrosine kinases of the Src family capable of phosphorylating the α-subunit of transducin, which is the heterotrimeric G protein involved in phototransduction, the purified tyrosine kinase was not capable of using transducin as a substrate. The cellular role of this retinal Src family member remains to be found.

Regulation of Discrete Functional Responses by Syk and Src Family Tyrosine Kinases in Human Neutrophils

Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs), as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1) involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.