The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here, we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility, and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signalling hub in a variety of cellular processes.

• Klíčová slova
• SH3 domain, Src, biochemistry, cell biology, cell transformation, chemical biology, invasiveness, mouse, phosphorylation, protein structure,
• MeSH
• fosforylace MeSH
• skupina kinas odvozených od src-genu * metabolismus   MeSH
• src homologní domény * MeSH
• tyrosin metabolismus   MeSH
• tyrosinkinasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• skupina kinas odvozených od src-genu * MeSH
• tyrosin MeSH
• tyrosinkinasy MeSH

Many patients with chronic myeloid leukemia in deep remission experience return of clinical disease after withdrawal of tyrosine kinase inhibitors (TKIs). This suggests signaling of inactive BCR-ABL, which allows the survival of cancer cells, and relapse. We show that TKI treatment inhibits catalytic activity of BCR-ABL, but does not dissolve BCR-ABL core signaling complex, consisting of CRKL, SHC1, GRB2, SOS1, cCBL, p85a-PI3K, STS1 and SHIP2. Peptide microarray and co-immunoprecipitation results demonstrate that CRKL binds to proline-rich regions located in C-terminal, intrinsically disordered region of BCR-ABL, that SHC1 requires pleckstrin homology, src homology and tyrosine kinase domains of BCR-ABL for binding, and that BCR-ABL sequence motif located in disordered region around phosphorylated tyrosine 177 mediates binding of three core complex members, i.e., GRB2, SOS1, and cCBL. Further, SHIP2 binds to the src homology and tyrosine kinase domains of BCR-ABL and its inositol phosphatase activity contributes to BCR-ABL-mediated phosphorylation of SHC1. Together, this study characterizes protein-protein interactions within the BCR-ABL core complex and determines the contribution of particular BCR-ABL domains to downstream signaling. Understanding the structure and dynamics of BCR-ABL interactome is critical for the development of drugs targeting integrity of the BCR-ABL core complex.

• Klíčová slova
• BCR–ABL, Chronic myeloid leukemia, Protein complex, Signaling,
• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• aminokyselinové motivy MeSH
• bcr-abl fúzní proteiny chemie   genetika   metabolismus   MeSH
• chronická myeloidní leukemie metabolismus   patologie   MeSH
• čipová analýza proteinů MeSH
• fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• pyrimidiny farmakologie   MeSH
• signální transdukce * účinky léků   MeSH
• src homologní domény MeSH
• transformující protein 1 obsahující src homologní doménu 2 metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• bcr-abl fúzní proteiny MeSH
• CRKL protein MeSH Prohlížeč
• fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy MeSH
• inhibitory proteinkinas MeSH
• INPPL1 protein, human MeSH Prohlížeč
• nilotinib MeSH Prohlížeč
• pyrimidiny MeSH
• transformující protein 1 obsahující src homologní doménu 2 MeSH

Attachment of stem leukemic cells to the bone marrow extracellular matrix increases their resistance to chemotherapy and contributes to the disease persistence. In chronic myelogenous leukemia (CML), the activity of the fusion BCR-ABL kinase affects adhesion signaling. Using real-time monitoring of microimpedance, we studied in detail the kinetics of interaction of human CML cells (JURL-MK1, MOLM-7) and of control BCR-ABL-negative leukemia cells (HEL, JURKAT) with fibronectin-coated surface. The effect of two clinically used kinase inhibitors, imatinib (a relatively specific c-ABL inhibitor) and dasatinib (dual ABL/SRC family kinase inhibitor), on cell binding to fibronectin is described. Both imatinib and low-dose (several nM) dasatinib reinforced CML cell interaction with fibronectin while no significant change was induced in BCR-ABL-negative cells. On the other hand, clinically relevant doses of dasatinib (100 nM) had almost no effect in CML cells. The efficiency of the inhibitors in blocking the activity of BCR-ABL and SRC-family kinases was assessed from the extent of phosphorylation at autophosphorylation sites. In both CML cell lines, SRC kinases were found to be transactivated by BCR-ABL. In the intracellular context, EC50 for BCR-ABL inhibition was in subnanomolar range for dasatinib and in submicromolar one for imatinib. EC50 for direct inhibition of LYN kinase was found to be about 20 nM for dasatinib and more than 10 µM for imatinib. Cells pretreated with 100 nM dasatinib were still able to bind to fibronectin and SRC kinases are thus not necessary for the formation of cell-matrix contacts. However, a minimal activity of SRC kinases might be required to mediate the increase in cell adhesivity induced by BCR-ABL inhibition. Indeed, active (autophosphorylated) LYN was found to localize in cell adhesive structures which were visualized using interference reflection microscopy.

• MeSH
• buněčná adheze účinky léků   MeSH
• chronická myeloidní leukemie patologie   MeSH
• dasatinib farmakologie   MeSH
• fibronektiny metabolismus   MeSH
• fosforylace účinky léků   MeSH
• imatinib mesylát farmakologie   MeSH
• kinetika MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• protinádorové látky farmakologie   MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• transport proteinů účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dasatinib MeSH
• fibronektiny MeSH
• imatinib mesylát MeSH
• protinádorové látky MeSH
• skupina kinas odvozených od src-genu MeSH

BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F). This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

• MeSH
• biologická evoluce * MeSH
• fosforylace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• src homologní domény * MeSH
• tyrosin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• tyrosin MeSH

Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligand-binding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein-tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas-/- mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells.

• MeSH
• fokální adheze metabolismus   MeSH
• fokální adhezní tyrosinkinasy metabolismus   MeSH
• fosforylace MeSH
• invazivní růst nádoru MeSH
• lidé MeSH
• molekuly buněčné adheze metabolismus   MeSH
• mutace MeSH
• myši MeSH
• nádorová transformace buněk MeSH
• nádorové buněčné linie MeSH
• pohyb buněk MeSH
• signální transdukce MeSH
• src homologní domény MeSH
• substrátový protein asociovaný s Crk chemie   genetika   metabolismus   MeSH
• transformované buněčné linie MeSH
• tyrosin metabolismus   MeSH
• tyrosinfosfatasa nereceptorového typu 12 metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fokální adhezní tyrosinkinasy MeSH
• molekuly buněčné adheze MeSH
• PTPN12 protein, human MeSH Prohlížeč
• substrátový protein asociovaný s Crk MeSH
• tyrosin MeSH
• tyrosinfosfatasa nereceptorového typu 12 MeSH
• zelené fluorescenční proteiny MeSH