Substrate conformational restriction and CD45-catalyzed dephosphorylation of tail tyrosine-phosphorylated Src protein.
ABSTRACT Hydrolysis of the tail phosphotyrosine in Src family members is catalyzed by the protein-tyrosine phosphatase CD45, activating Src family-related signaling pathways. Using purified recombinant phospho-Src (P-Src) (amino acid residues 83-533) and purified recombinant CD45 catalytic (cytoplasmic) domain (amino acid residues 565-1268), we have analyzed the kinetic behavior of dephosphorylation. A time course of phosphatase activity showed the presence of a burst phase. By varying the concentration of P-Src, it was shown that the amplitude of this burst phase increased linearly with respect to P-Src concentration. Approximately 2% of P-Src was shown to be rapidly dephosphorylated followed by a slower linear phase. A P-Src protein substrate containing a functional point mutation in the Src homology domain 2 (SH2) led to more rapid dephosphorylation catalyzed by CD45, and this reaction showed only a single linear kinetic phase. These results were interpreted in terms of a model in which P-Src exists in a relatively slow dynamic equilibrium between "closed" and "open" conformational forms. Combined mutations in the SH2 and SH3 domain or the addition of an SH3 domain ligand peptide enhanced the accessibility of P-Src to CD45 by biasing P-Src to a more open form. Consistent with this model, a phosphotyrosine peptide that behaved as an SH2 domain binding ligand showed approximately 100-fold greater affinity for unphosphorylated Src versus P-Src. Surprisingly, P-Src possessing combined SH3 and SH2 functional inactivating point mutations was dephosphorylated by CD45 more slowly compared with P-Src completely lacking SH3 and SH2 domains. Additional data suggest that the SH3 and SH2 domains can inhibit accessibility of the P-Src tail to CD45 by interactions other than direct phosphotyrosine binding by the SH2 domain. Taken together, these results suggest how activation of Src family member signaling pathways by CD45 may be influenced by the presence or absence of ligand interactions remote from the tail.
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ABSTRACT: The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380-385) and these phosphorylations are proposed to induce a reduction in PTEN's plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN's regulation and suggest pharmacologic approaches for direct PTEN activation. DOI:http://dx.doi.org/10.7554/eLife.00691.001.eLife Sciences 07/2013; 2:e00691. · 8.52 Impact Factor
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ABSTRACT: Src family kinases such as Lyn are important signaling intermediaries, relaying and modulating different inputs to regulate various outputs, such as proliferation, differentiation, apoptosis, migration and metabolism. Intriguingly, Lyn can mediate both positive and negative signaling processes within the same or different cellular contexts. This duality is exemplified by the B-cell defect in Lyn-/- mice in which Lyn is essential for negative regulation of the B-cell receptor; conversely, B-cells expressing a dominant active mutant of Lyn (Lynup/up) have elevated activities of positive regulators of the B-cell receptor due to this hyperactive kinase. Lyn has well-established functions in most haematopoietic cells, viz. progenitors via influencing c-kit signaling, through to mature cell receptor/integrin signaling, e.g. erythrocytes, platelets, mast cells and macrophages. Consequently, there is an important role for this kinase in regulating hematopoietic abnormalities. Lyn is an important regulator of autoimmune diseases such as asthma and psoriasis, due to its profound ability to influence immune cell signaling. Lyn has also been found to be important for maintaining the leukemic phenotype of many different liquid cancers including acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML) and B-cell lymphocytic leukaemia (BCLL). Lyn is also expressed in some solid tumors and here too it is establishing itself as a potential therapeutic target for prostate, glioblastoma, colon and more aggressive subtypes of breast cancer. LAY ABSTRACT: To relay information, a cell uses enzymes that put molecular markers on specific proteins so they interact with other proteins or move to specific parts of the cell to have particular functions. A protein called Lyn is one of these enzymes that regulate information transfer within cells to modulate cell growth, survival and movement. Depending on which type of cell and the source of the information input, Lyn can positively or negatively regulate the information output. This ability of Lyn to be able to both turn on and turn off the relay of information inside cells makes it difficult to fully understand its precise function in each specific circumstance. Lyn has important functions for cells involved in blood development, including different while blood cells as well as red blood cells, and in particular for the immune cells that produce antibodies (B-cells), as exemplified by the major B-cell abnormalities that mice with mutations in the Lyn gene display. Certain types of leukaemia and lymphoma appear to have too much Lyn activity that in part causes the characteristics of these diseases, suggesting it may be a good target to develop new anti-leukaemia drugs. Furthermore, some specific types, and even specific subtypes, of solid cancers, e.g. prostate, brain and breast cancer can also have abnormal regulation of Lyn. Consequently, targeting this protein in these cancers could also prove to be beneficial.Cell Communication and Signaling 07/2012; 10(1):21. · 4.67 Impact Factor
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ABSTRACT: The association of the SH3 (Src homology 3) domain of SFKs (Src family kinases) with protein partners bearing proline-rich motifs has been implicated in the regulation of SFK activity, and has been described as a possible mechanism of relocalization of SFKs to subcellular compartments. We demonstrate in the present study for the first time that p13, an accessory protein encoded by the HTLV-1 (human T-cell leukaemia virus type 1), binds the SH3 domain of SFKs via its C-terminal proline-rich motif, forming a stable heterodimer that translocates to mitochondria by virtue of its N-terminal mitochondrial localization signal. As a result, the activity of SFKs is dramatically enhanced, with a subsequent increase in mitochondrial tyrosine phosphorylation, and the recognized ability of p13 to insert itself into the inner mitochondrial membrane and to perturb the mitochondrial membrane potential is abolished. Overall, the present study, in addition to confirming that the catalytic activity of SFKs is modulated by interactors of their SH3 domain, leads us to hypothesize a general mechanism by which proteins bearing a proline-rich motif and a mitochondrial localization signal at the same time may act as carriers of SFKs into mitochondria, thus contributing to the regulation of mitochondrial functions under various pathophysiological conditions.Biochemical Journal 07/2011; 439(3):505-16. · 4.78 Impact Factor