Transmembrane Phosphoprotein Cbp Positively Regulates the Activity of the Carboxyl-terminal Src Kinase, Csk

Abstract

Csk (carboxyl-terminal Src kinase) is a cytoplasmic tyrosine kinase that phosphorylates a critical tyrosine residue in each of the Src family kinases (SFKs) to inhibit their activities. Recently, we identified a transmembrane protein, Cbp (Csk-binding protein), that, when phosphorylated, can recruit Csk to the membrane where the SFKs are located. The Cbp-mediated relocation of Csk to the membrane may play a role in turning off the signaling events initiated by SFKs. To further characterize the Csk-Cbp interaction, we have generated a reconstituted system using soluble, highly purified proteins. Csk and phosphorylated Cbp were co-purified as a large protein complex consisting of at least four Csk⅐Cbp units. The addition of the phosphorylated, but not nonphosphorylated, Cbp to an in vitro assay stimulated Csk activity toward Src. Csk was also activated by a phosphopeptide containing the tyrosine in Cbp that binds to Csk (Tyr-314). Kinetic analysis revealed that Cbp or the phosphopeptide induced up to a 6-fold reduction in the K m for Src, indicating that the Csk⅐Cbp complex has a greater affinity for Src than free Csk. These findings suggest that Cbp is involved in the regulation of SFKs not only by relocating Csk to the membrane but also by directly activating Csk.The Src family kinases (SFKs) 1 are nonreceptor protein tyrosine kinases (PTKs) that are associated with the inner surface of plasma membrane through their fatty-acylated amino termini (1). SFKs are known to act as molecular switches that regulate a variety of cellular events, including cell growth and division, cell attachment and movement, differentiation, survival, or death (2). SFKs are ordinarily present in an inactive state in which the phosphorylated carboxyl-terminal regulatory tyrosine binds to its own SH2 domain (3). In response to an external stimulus, an SFK is activated through dephosphorylation of the carboxyl-terminal tyrosine or through binding to another protein that displaces the intramolecular interaction. The phosphorylation of the regulatory tyrosine of SFK is known to be catalyzed by another PTK, Csk (4, 5). In contrast, the phosphatases that activate SFKs have not yet been positively identified, although some candidate molecules have been proposed (6, 7). To understand the regulation of SFKs, it is essential to clarify the regulation mechanism controlling the phosphorylation and dephosphorylation of the critical carboxylterminal tyrosine.Csk is a cytoplasmic PTK consisting of an SH3, an SH2, and a kinase domain. Because it lacks an amino-terminal acylation signal and a carboxyl-terminal tyrosine, the regulatory mechanisms of Csk itself have remained unknown. A line of evidence has suggested that the SH2 and/or SH3 domain of Csk is essential for SFK regulation (8, 9). The relocation of Csk to the membrane, specifically to regions where SFKs are active, was also observed (10). In addition, a membrane-targeted form of Csk, containing the myristoylation signal of Src, more actively suppressed SFK functions (11). These facts suggeste...

Cutting Edge: Fyn Is Essential for Tyrosine Phosphorylation of Csk-Binding Protein/Phosphoprotein Associated with Glycolipid-Enriched Microdomains in Lipid Rafts in Resting T Cells

“…As shown in Fig. 3B, Cbp IPs from normal thymocytes were capable of phosphorylating poly EY efficiently, and this phosphorylation was almost completely blocked by the addition of Cbp-4 phosphopeptide which contains Tyr 314 and is known to compete with Cbp-Csk interaction (12). Another phosphopeptide, Cbp-2, which cannot interfere with the binding of Cbp to Csk was not able to inhibit the phosphorylation of poly EY.…”

“…Once recruited to rafts and activated by Cbp, Csk would inhibit the kinase activity of Lck through phosphorylation of an Lck C-terminal tyrosine to keep resting T cells in a quiescent state. The Csk-Cbp complex has been shown to have a greater affinity for Src than free Csk (12). Although Fyn itself could be a target of Csk in rafts, it has been demonstrated that the kinase activity of Fyn is relatively difficult to be suppressed by Csk compared with those of other Src family PTKs (16).…”

“…Cbp IPs was suspended in KB with 4 g of poly EY and 10 Ci [␥-32 P]ATP. To block Cbp-Csk interaction, Cbp-4 (ISAMYSSVNK) or Cbp-2 (EDCLYETVKE) phosphopeptides were added to cell lysates before immunoprecipitation with anti-Cbp Abs as previously described (12). The activity was visualized and quantified using a BAS-1500 Bioimage Analyzer (Fuji Photo Film, Tokyo, Japan).…”

“…These results is able to inhibit Cbp-Csk interaction, while Cbp-2 is not. Csk was purified from infected insect cells as described previously (12). C, Tyr 505 phosphorylation and the total amount of Lck in the raft fraction from normal B6 or Fyn Ϫ/Ϫ thymocytes were analyzed by anti-phospho-Lck (Y505) and anti-Lck blotting, respectively.…”

Selected glimpses into the activation and function of Src kinase

“…Cbp/ PAG binds to and recruits CSK to the plasma membrane by virtue of a tyrosine phosphorylation site on Cbp/PAG and the SH2 domain in CSK. Binding of Cbp/PAG to CSK also appears to activate CSK (Takeuchi et al, 2000). The tyrosine kinase responsible for the phosphorylation of Cbp/PAG is currently unknown.…”