Beta2 integrin-dependent phosphorylation of protein-tyrosine kinase Pyk2 stimulated by tumor necrosis factor alpha and fMLP in human neutrophils adherent to fibrinogen.
(49/4322)
Tumor necrosis factor alpha and fMLP can activate a broad range of cellular functions in neutrophils adherent to biological surfaces. These functions are mediated by integrins and involve the activation of tyrosine kinases. Here, we report that Pyk2, a member of the focal adhesion kinase family, was present in human neutrophils and was rapidly phosphorylated and activated following tumor necrosis factor alpha and fMLP stimulation in an adhesion-dependent manner. Tyrosine phosphorylation of Pyk2 was attenuated by beta2 integrin blocking with specific antibodies. The tyrosine phosphorylation of Pyk2 was downstream of protein kinases Lyn, Syk and protein kinase C and cytoskeletal organization. The activation of Pyk2 may play a role in adhesion/cytoskeleton-associated neutrophils function. (+info)
ErbB receptor-induced activation of stat transcription factors is mediated by Src tyrosine kinases.
(50/4322)
Epidermal growth factor (EGF) binding to its receptor, ErbB1, triggers various signal transduction pathways, one of which leads to the activation of signal transducer and activator of transcription (Stat) factors. The mechanism underlying ErbB1-induced Stat activation and whether Stats are downstream targets of other ErbB receptors have not been explored. In this report we show that ErbB2, ErbB3, and ErbB4 do not potentiate Stat5 phosphorylation by EGF. However, neu differentiation factor-induced heterodimers of ErbB2 and ErbB4 activated Stat5. In A431 cells, Stat1, Stat3, and Stat5, were constitutively complexed with ErbB1 and rapidly phosphorylated on tyrosine in response to EGF. Neither mutation of the conserved tyrosine residue (Tyr694) nor inactivation of the Stat5a SH2 domain disrupted this association. However, an intact SH2 domain was necessary for EGF-induced Stat5a phosphorylation. In contrast to prolactin, which induced only Tyr694 phosphorylation of Stat5a, EGF promoted phosphorylation on Tyr694 and additional tyrosine residue(s). Janus kinases (Jaks) were also constitutively associated with ErbB receptors and were phosphorylated in response to EGF-related ligands. However, we provide evidence that EGF- and neu differentiation factor-induced Stat activation are dependent on Src but not Jak kinases. Upon EGF stimulation, c-Src was rapidly recruited to Stat/ErbB receptor complexes. Pharmacological Src kinase inhibitors and a dominant negative c-Src ablated both Stat and Jak tyrosine phosphorylation. However, dominant negative Jaks did not affect EGF-induced Stat phosphorylation. Taken together, the experiments establish two independent roles for Src kinases: (i) key molecules in ErbB receptor-mediated Stat signaling and (ii) potential upstream regulators of Jak kinases. (+info)
CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.
(51/4322)
Ligation of the B cell Ag receptor (BCR) induces cellular activation by stimulating Src-family protein tyrosine kinases (PTKs) to phosphorylate members of the BCR complex. Subsequently, Src-family PTKs, particularly Lyn, are proposed to phosphorylate and bind CD19, a cell-surface costimulatory molecule that regulates mature B cell activation. Herein, we show that B cells from CD19-deficient mice have diminished Lyn kinase activity and BCR phosphorylation following BCR ligation. Tyrosine phosphorylation of other Src-family PTKs was also decreased in CD19-deficient B cells. In wild-type B cells, CD19 was constitutively complexed with Vav, Lyn, and other Src-family PTKs, with CD19 phosphorylation and its associations with Lyn and Vav increased after BCR ligation. Constitutive CD19/Lyn/Vav complex signaling may therefore be responsible for the establishment of baseline signaling thresholds in B cells before Ag receptor ligation, in addition to accelerating signaling following BCR engagement or other transmembrane signals. In vitro kinase assays using purified CD19 and purified Lyn revealed that the kinase activity of Lyn was significantly increased when coincubated with CD19. Thus, constitutive and induced CD19/Lyn complexes are likely to regulate basal signaling thresholds and BCR signaling by amplifying the kinase activity of Lyn and other Src-family PTKs. These in vivo and in vitro findings demonstrate a novel mechanism by which CD19 regulates signal transduction in B lymphocytes. The absence of this CD19/Src-family kinase amplification loop may account for the hyporesponsive phenotype of CD19-deficient B cells. (+info)
Fyn membrane localization is necessary to induce the constitutive tyrosine phosphorylation of Sam68 in the nucleus of T lymphocytes.
(52/4322)
A close relationship between Sam68, a tyrosine and proline-rich RNA-binding protein, and Src protein tyrosine kinases (PTK) has already been established, also in T lymphocytes. A constitutive phosphorylation of the molecule has also been documented in various transformed T cells, which probably reflects an increased expression of PTK of the Src family. Using the hybridoma T cell line, T8.1, or Jurkat T cells, we investigated the respective contribution of the two Src kinases Fyn and Lck, expressed in T cells, in this phenomenon. By overexpressing the two proteins, we show that the constitutive phosphorylation of Sam68 in vivo directly correlates with cellular Fyn levels, but not with Lck expression, despite the capacity of the PTK to strongly phosphorylate the molecule in vitro. Overexpressed Fyn is mainly localized at the cell membrane. We find that Sam68 phosphorylation, including in the nuclear fraction in which the molecule is predominantly expressed, is lost with a delocalized Fyn mutant deleted of its N-terminal membrane-anchoring domain. Finally, we demonstrate, using a construct encoding a Sam68 molecule without its nuclear localization signal, that nuclear expression of Sam68 is not required for phosphorylation. We conclude that the constitutive phosphorylation of Sam68 in T cells is a Fyn-dependent process occurring in a cell-membrane compartment from which phospho-Sam68 molecules can thereafter accumulate into the nucleus. (+info)
Transcriptional up-regulation of matrix metalloproteinase-9 expression during spontaneous epithelial to neuroblast phenotype conversion by SK-N-SH neuroblastoma cells, involved in enhanced invasivity, depends upon GT-box and nuclear factor kappaB elements.
(53/4322)
Spontaneous epithelial (S) to neuroblast (N) conversion enhanced the capacity of SK-N-SH neuroblastoma (NB) cells to invade reconstituted basement membrane in vitro. This involved a switch to matrix metalloproteinase (MMP) activity, in particular MMP-9, and was associated with the induction of MMP-9 expression. N-type-specific MMP-9 expression was herbimycin A inhibitable tyrosine kinase (possibly c-src) dependent and was regulated transcriptionally through GT-box (-52), and nuclear factor kappaB (NFkappaB; -600) elements within the MMP-9 gene. GT-box function was associated with elevated levels of specific nuclear GT-box binding complexes in N-type cells. NFkappaB function was associated with specific p50- and p65-containing nuclear NFkappaB binding complex(es). No function could be attributed to the proximal AP-1 (-79) element, and minimal function was attributed to the SP-1 (-560), ets (-540), or distal AP-1 (-533) elements. This was despite elevated levels of specific junD/fra-1 containing proximal AP-1 element binding complex(es) in N-type cells. Our data highlight a pivotal role for the GT-box, in concert with the NFkappaB element, in the transcriptional up-regulation of MMP-9 expression during spontaneous S to N phenotype conversion by SK-N-SH cells involved in enhanced basement membrane invasivity. (+info)
The tyrosine kinases Syk and Lyn exert opposing effects on the activation of protein kinase Akt/PKB in B lymphocytes.
(54/4322)
The protein kinase Akt/PKB is a crucial regulator of cell survival in response to mitogenic signals. The increased kinase activity of v-akt, an oncogenic form of Akt/PKB, causes mouse T cell lymphoma, and overexpression of Akt/PKB is associated with progression of several tumor types in human. In this study, we demonstrate that ligation of B cell antigen receptor (BCR) leads to activation of Akt/PKB in B lymphocytes. BCR-induced activation of Akt/PKB required the tyrosine kinase Syk, which was not previously known to regulate Akt/PKB. In contrast, BCR crosslinking of Lyn-deficient B cells resulted in markedly enhanced hyperphosphorylation and activation of Akt/PKB compared with wild-type B cells, indicating that this Src-family kinase acts as an endogenous antagonist of BCR-induced Akt/PKB activation. Lyn inhibited Akt/PKB additively with an okadaic acid-sensitive endogenous phosphatase(s). Expression of exogenous Lyn in mutant cells restored normal BCR-induced phosphorylation of Akt/PKB. Negative regulation of Akt/PKB by Lyn was not dependent on the protein phosphatases SHP-1, SHP-2, or SHIP. Our results show that Lyn provides a mechanism for negative regulation and opposes the effect of Syk on BCR-mediated activation of Akt/PKB. Deregulation of Akt/PKB correlates with the hyperresponsiveness of B cells from Lyn-deficient mice stimulated by BCR crosslinking and may contribute to the autoimmune syndrome that develops in Lyn-deficient animals. (+info)
The angiotensin II-dependent association of Jak2 and c-Src requires the N-terminus of Jak2 and the SH2 domain of c-Src.
(55/4322)
The binding of angiotensin II (Ang II) to AT1 is known to increase the kinase activity of several nonreceptor tyrosine kinases including Jak2 and c-Src. In the present study, we demonstrate that treatment of vascular smooth muscle cells with Ang II results in a rapid and transient association of Jak2 and c-Src. This association is dependent on a catalytically active Jak2 kinase, because it is blocked both by pharmacological means and by the inability of a catalytically inactive Jak2 to associate with c-Src. c-Src bound tyrosine phosphorylated Jak2 but was unable to bind an equal amount of unphosphorylated Jak2 protein, indicating that the SH2 domain of c-Src mediates this association. In vivo studies indicated that c-Src binds the N-terminus of Jak2 as expression of a Jak2 molecule lacking the initial 240 amino acids, including 16 tyrosines, and was unable to bind c-Src. Lastly, using transiently transfected COS-7 cells, we found that Ang II treatment induced an association between c-Src and wild-type Jak2 but not between c-Src and the Jak2 molecule that lacks the initial 240 amino acids. Thus, our data suggest that in addition to increasing the kinase activities Jak2 and c-Src, treatment of cells with Ang II results in the physical association of Jak2 and c-Src; an association that is mediated by the SH2 domain of c-Src and the N-terminus of Jak2. (+info)
Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase.
(56/4322)
In the central nervous system, myelination of axons occurs when oligodendrocyte progenitors undergo terminal differentiation and initiate process formation and axonal ensheathment. Although it is hypothesized that neuron-oligodendrocyte contact initiates this process, the molecular signals are not known. Here we find that Fyn tyrosine kinase activity is upregulated very early during oligodendrocyte progenitor cell differentiation. Concomitant with this increase is the appearance of several tyrosine phosphorylated proteins present only in differentiated cells. The increased tyrosine kinase activity is specific to Fyn, as other Src family members are not active in oligodendrocytes. To investigate the function of Fyn activation on differentiation, we used Src family tyrosine kinase inhibitors, PP1 and PP2, in cultures of differentiating oligodendrocyte progenitors. Treatment of progenitors with these compounds prevented activation of Fyn and reduced process extension and myelin membrane formation. This inhibition was reversible and not observed with related inactive analogues. A similar effect was observed when a dominant negative Fyn was introduced in progenitor cells. These findings strongly suggest that activation of Fyn is an essential signaling component for the morphological differentiation of oligodendrocytes. (+info)