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.
"N-terminal sequencing identified several of these cleavage positions (Figure 4B). Alkaline phosphatase sensitivity and western blotting was used to assess the exposure of the C-terminal 380–385 phospho cluster in 4p-PTEN (Wang et al., 2002). The half-life of alkaline phosphatase-mediated dephosphorylation of 4p-PTEN under the conditions used was found to be ∼60 min (Figure 4C and Figure 4— figure supplement 2A). "
[Show abstract][Hide abstract] ABSTRACT: eLife digest
PTEN is an enzyme that is found in almost every tissue in the body, and its job is to stop cells dividing. If it fails to perform this job, the uncontrolled proliferation of cells can lead to the growth of tumors. PTEN stops cells dividing by localizing at the plasma membrane of a cell and removing a phosphate group from a lipid called PIP3: this sends a signal, via the PI3K pathway, that suppresses the replication and survival of cells.
Three regions of PTEN are thought to be central to its biological functions: one of these regions, the phosphatase domain, is directly responsible for removing a phosphate group from the lipid PIP3; a second region, called the C2 domain, is known to be critical for PTEN binding to the cell membrane; however, the role of third region, called the C-terminal domain, is poorly understood.
Many proteins are regulated by the addition and removal of phosphate groups, and PTEN is no exception. In particular, it seems as if the addition of phosphate groups to four amino acid residues in the C-terminal domain can switch off the activity of PTEN, but the details of this process have been elusive.
Now, Bolduc et al. have employed a variety of biochemical and biophysical techniques to explore this process, finding that the addition of the phosphate groups reduced PTEN’s affinity for the plasma membrane. At the same time, interactions between the C-terminal and C2 domains of the PTEN cause the shape of the enzyme to change in a way that ‘buries’ the residues to which the phosphate groups have been added.
In addition to offering new insights into PTEN, the work of Bolduc et al. could help efforts to identify compounds with clinical anti-cancer potential.
"Lyn’s SH3 domain can bind an intramolecular proline-motif situated between the SH2 and kinase domains (hinge region), helping generate a stabilized inactive kinase confirmation. Activation of Lyn involves dephosphorylation of the C-terminal tyrosine (Y508) by phosphatases such as CD45  and SHP-2 , as well as through interactions with SH2 and/or SH3 domain binding motifs, which compete with Lyn's own SH3/SH2 intramolecular interaction sites, thus releasing the auto-inhibitory configuration of the kinase domain. Lyn can then trans-autophosphorylate within the activation loop (Y397) to generate a highly active enzyme. "
[Show abstract][Hide abstract] 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.
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. DOI:10.1186/1478-811X-10-21 · 3.38 Impact Factor
"However , there are also reports that illustrate how CD45 can also be a negative regulator of SFK activity and signalling . It is interesting that the ability of CD45 to catalytically attack the C-terminal SFK site is altered by SFK structure and its interaction with other molecules , thus showing again the importance of SFK complex formation in regulating their activity. While phosphorylation/dephosphorylation of the C-terminal tyrosine has a mild effect on intrinsic kinase activity, the phosphorylation status of the A-loop motif has a substantial effect upon SFK enzyme catalysis. "
[Show abstract][Hide abstract] ABSTRACT: While the Src family of protein tyrosine kinases (SFK), and the main ancillary molecules involved in their regulation, have been studied for many years, the details of their interplay are not fully understood and thus remain under active investigation. Additionally, new players that coordinate their regulation and direct their signalling cascades are also being uncovered, shedding new light on the complexity of these signalling networks. Through the utilization of novel interaction assays, several new interconnecting mediators that are helping to show the elegance of Src family kinase regulation have been discovered. This review outlines SFK regulation, the discovery of the Csk binding protein (Phosphoprotein Associated with Glycosphingolipid-enriched microdomains, Cbp/PAG), and its role in regulating SFK kinase activity status, as well as protein levels. Further, details of the methods used to identify this dual mode of regulation can be applied to delineate the full gamut of SH2/SH3-directed SFK pathways and, indeed, those of any tyrosine kinase. Using Lyn as a model SFK, we and others have shown that Cbp recruits negative regulators of COOH-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk) after Lyn is activated and bound to Cbp. Lyn phosphorylates Cbp on multiple tyrosine residues, including two that can bind Lyn's SH2 domain with high affinity. Lyn also phosphorylates Y314, which recruits Csk/Ctk to phosphorylate Lyn at its Y508 negative site, allowing an inactive conformation to form. However, the pY508 site has a low affinity for Lyn's SH2 domain, while the Cbp sites have high affinity. Thus, until these Cbp sites are dephosphorylated, Lyn can remain active. Intriguingly, phosphorylated Y314 also binds the suppressor of cytokine signalling 1 (SOCS1), resulting in elevated ubiquitination and degradation of Lyn. Thus, a single phosphotyrosine residue within Cbp co-ordinates a two-phase process involving distinct negative regulatory pathways that allow inactivation, followed by degradation, of SFKs.
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