Regulation of DNA-dependent protein kinase activity in leukemic cells

The DNA-dependent protein kinase (DNA-PK) complex is composed of a catalytic (DNA-PKcs), and a regulatory subunit (Ku70/Ku86 heterodimer). The expression and function of DNA-PK subunits was investigated in purified blood lymphocytes obtained from patients with chronic lymphocytic leukemia (CLL) either refractory to chemotherapy or untreated. Variations in DNA-PK activity were found amongst CLL samples by comparison to human cell lines. It was noticeable that the low DNA-PK activity was associated with samples from untreated patients that exhibited a sensitivity phenotype, determined in vitro, to the radiomimetic agent neocarcinostatin by comparison to samples from refractory patients. The regulation in DNA-PK activity was associated with Ku heterodimer expression while DNA-PKcs was unaffected. Moreover, the presence of an altered form of the Ku86 subunit was identified in samples with low DNA-PK activity. These results suggest a regulation process of the DNA-PK activity in fresh human cells.     

The AMPA receptor interacts with and signals through the protein tyrosine kinase Lyn

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. The ionotropic glutamate receptors are classified into two groups, NMDA (N-methyl-D-aspartate) receptors and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors. The AMPA receptor is a ligand-gated cation channel that mediates the fast component of excitatory postsynaptic currents in the central nervous system. Here we report that AMPA receptors function not only as ion channels but also as cell-surface signal transducers by means of their interaction with the Src-family non-receptor protein tyrosine kinase Lyn. In the cerebellum, Lyn is physically associated with the AMPA receptor and is rapidly activated following stimulation of the receptor. Activation of Lyn is independent of Ca2+ and Na+ influx through AMPA receptors. As a result of activation of Lyn, the mitogen-activated protein kinase (MAPK) signalling pathway is activated, and the expression of brain-derived neurotrophic factor (BDNF) messenger RNA is increased in a Lyn-kinase-dependent manner. Thus, AMPA receptors generate intracellular signals from the cell surface to the nucleus through the Lyn-MAPK pathway, which may contribute to synaptic plasticity by regulating the expression of BDNF.     

Regulation of T-cell antigen receptor signalling by Syk tyrosine protein kinase

T-cell antigen receptor (TCR) signalling has been shown to involve two classes of tyrosine protein kinases: the Src-related kinases p56(lck) and p59(fyr), and the Zap-70/Syk family kinases. Lck and FynT are postulated to initiate TCR-triggered signal transduction by phosphorylating the CD3 and zeta subunits of the TCR complex. This modification permits the recruitment of Zap-70 and Syk, which are presumed to amplify the TCR-triggered signal, by phosphorylating additional intracellular proteins. While Zap-70 is expressed in all T cells, Syk is present in thymocytes and mature T-cell populations such as intraepithelial gammadelta T cells and naive alphabeta T cells. To better understand the role of Syk in these cells, its impact on the physiology of an antigen-specific T-cell line was tested. Our results showed that compared to Zap-70 alone, Syk was a strong positive regulator of antigen receptor-induced signals in BI-141 cells. Surprisingly, they indicated that, like Src family kinases, Syk augmented TCR-triggered tyrosine phosphorylation of CD3/zeta. Syk, but not Zap-70 alone, could also stimulate tyrosine phosphorylation of a zeta-bearing chimera in transiently transfected Cos-1 cells. Finally, evidence was provided that Syk has the capacity to directly phosphorylate a zeta-derived peptide in vitro. These findings suggested that Syk may have a unique role in T cells, as a consequence of its ability to efficiently phosphorylate multiple components of the TCR signalling cascade. Furthermore, they raised the possibility that Syk can regulate the initiation of TCR signalling, by promoting phosphorylation of the immunoreceptor tyrosine-based activation motifs of the TCR complex.     

Inactivation of DNA-dependent protein kinase by protein kinase Cdelta: implications for apoptosis

Protein kinase Cdelta (PKCdelta) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCdelta in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCdelta (PKCdelta CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCdelta cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCdelta associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCdelta CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCdelta CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCdelta CF. These findings support the hypothesis that functional interactions between PKCdelta and DNA-PK contribute to DNA damage-induced apoptosis.     

Interaction of cyclin-dependent kinase 2 and the Lyn tyrosine kinase in cells treated with 1-beta-D-arabinofuranosylcytosine

The cyclin dependent kinase 2 (Cdk2) is required for initiation and progression of DNA replication. Activation of Cdk2 involves binding to cyclin E or cyclin A and dephosphorylation of Tyr15. The present studies demonstrate that treatment of U-937 cells with 1-beta-D-arabinofuranosylcytosine (ara-C) is associated with tyrosine phosphorylation of Cdk2 and inhibition of Cdk2 activity. The results also demonstrate that Cdk2 directly associates with the Src-like tyrosine kinase Lyn as a consequence of ara-C-treatment. Confocal microscopy studies show that Lyn is detectable in the nucleus and that it colocalises with Cdk2. Subcellular fractionation and coimmunoprecipitation studies further demonstrate nuclear binding of Lyn and Cdk2. We also show that Lyn phosphorylates Tyr15 of Cdk2 and that incubation of Lyn with Cdk2 results in inhibition of Cdk2 activity. These findings suggest that the association of Lyn and Cdk2 in ara-C-treated cells may contribute to regulation of Cdk2-dependent cell cycle checkpoints.     

Regulation of Bcr-Abl-induced SAP kinase activity and transformation by the SHPTP1 protein tyrosine phosphatase

The oncogenic Bcr-Abl variant of the c-Abl tyrosine kinase transforms cells by a mechanism dependent on activation of the stress-activated protein kinase (SAPK). Other work has shown that c-Abl interacts with the SHPTP1 protein tyrosine phosphatase in induction of SAPK activity by genotoxic stress. The present studies demonstrate that Bcr-Abl binds constitutively to SHPTP1. We show that Bcr-Abl phosphorylates SHPTP1 on C-terminal Y536 and Y564 sites. The functional significance of the Bcr-Abl/SHPTP1 interaction is supported by the finding that SHPTP1 regulates Bcr-Abl-induced SAPK activity. Importantly, SHPTP1 also decreases Bcr-Abl-dependent transformation of fibroblasts. These findings indicate that SHPTP1 functions as a tumor suppressor in cells transformed by Bcr-Abl.     

Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1, 5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.     

Regulation of the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 3

pp72syk is essential for development and function of several hematopoietic cells, and it becomes activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to study Syk activation by the platelet integrin, alpha IIb beta 3. As in platelets, Syk underwent tyrosine phosphorylation and activation during CHO cell adhesion to alpha IIb beta 3 ligands, including fibrinogen. This involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited two novel features. Firstly, unlike alpha IIb beta 3-mediated activation of pp125FAK, Syk activation could be triggered by the binding of soluble fibrinogen and abolished by truncation of the alpha IIb or beta 3 cytoplasmic tail, and it was resistant to inhibition by cytochalasin D. Secondly, it did not require phosphorylated ITAMs since it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains. These studies demonstrate that Syk is a proximal component in alpha IIb beta 3 signaling and is regulated as a consequence of intimate functional relationships with the alpha IIb beta 3 cytoplasmic tails and with Src or a closely related kinase. Furthermore, there are fundamental differences in the activation of Syk by alpha IIb beta 3 and immune response receptors, suggesting a unique role for integrins in Syk function.     

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells

The effect of protein tyrosine kinases (PTKs) on L-type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L-type channels were measured in the perforated patch-clamp technique and identified by using the L-type calcium channel opener Bay K8644 (10(-6) M). Application of the PTK blockers genistein (5 x 10(-6) M) or lavendustin A (5 x 10(-6) M) led to a decrease of L-type currents. The inactive genistein analog daidzein (10(-5) M) showed no effect on calcium channels. Intracellular application of pp60(c-src) (30 U/ml) via the patch-pipette during the conventional whole-cell configuration led to an increase of L-type currents. The protein kinase A and protein kinase G blocker H9 (10(-6) M) showed no effect on L-type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10(-5) M) reduced the L-type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5 x 10(-5) M) for PKC inhibition led to a fast rundown of L-type current amplitudes. Intracellularly applied myristoylated PKC substrate (10(-4) M) together with pp60(c-src) showed no effect on L-type current. Up-regulation of PKC by 10(-6) M phorbol-12-myristate-13-acetate (PMA) had no effect on the L-type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L-type currents. Intracellular application of pp60(c-src) in PMA-treated cells led to a reduction of L-type currents. We conclude that in the resting cell, PTK and PKC regulate L-type calcium channels in an additive manner. L-type channels appeared as a site of integration of PTK activation and of PKC-dependent pathways. The activity of PKC determines whether PTK decreases or increases L-type channel activity.     

Regulation of fibroblast motility by the protein tyrosine phosphatase PTP-PEST

The protein tyrosine phosphatase PTP-PEST is a cytosolic enzyme that displays a remarkable degree of selectivity for tyrosine-phosphorylated p130(Cas) as a substrate, both in vitro and in intact cells. We have investigated the physiological role of PTP-PEST using Rat1 fibroblast-derived stable cell lines that we have engineered to overexpress PTP-PEST. These cell lines exhibit normal levels of tyrosine phosphorylation of the majority of proteins but have significantly lower levels of tyrosine phosphorylation of p130(Cas) than control cells. Initial cellular events occurring following integrin-mediated attachment to fibronectin (cell attachment and spreading) are essentially unchanged in cells overexpressing PTP-PEST; similarly, the extent and time course of mitogen-activated protein kinase activation in response to integrin engagement is unchanged. In contrast, the reduced phosphorylation state of p130(Cas) is associated with a considerably reduced rate of cell migration and a failure of cells overexpressing PTP-PEST to accomplish the normally observed redistribution of p130(Cas) to the leading edge of migrating cells. Furthermore, cells overexpressing PTP-PEST demonstrate significantly reduced levels of association of p130(Cas) with the Crk adaptor protein. Our results suggest that one physiological role of PTP-PEST is to dephosphorylate p130(Cas), thereby controlling tyrosine phosphorylation-dependent signaling events downstream of p130(Cas) and regulating cell migration.     

Cryo-EM imaging of the catalytic subunit of the DNA-dependent protein kinase

The DNA-dependent protein kinase (DNA-PK) plays an important role in mammalian DNA double-strand break repair and immunoglobulin gene rearrangement. The DNA-PK holoenzyme is activated by assembly at DNA ends and is comprised of DNA-PKcs, a 460 kDa protein kinase catalytic subunit, and Ku, a 70 kDa/80 kDa heterodimeric DNA-targeting component. We have solved the three-dimensional structure of DNA-PKcs to approximately 21 A resolution by analytically combining images of nearly 9500 individual particles extracted from cryo-electron micrographs. The DNA-PKcs protein has an open, pseudo 2-fold symmetric structure with a gap separating a crown-shaped top from a rounded base. Columns of density are observed to protrude into the gap from both the crown and the base. Measurements of the enclosed volume indicate that the interior of the protein is largely hollow. The structure of DNA-PKcs suggests that its association with DNA may involve the internalization of double-stranded ends.     

Regulation of Schizosaccharomyces pombe Wee1 tyrosine kinase

Wee1 tyrosine kinase regulates mitosis by carrying out the inhibitory tyrosine 15 phosphorylation of Cdc2 M-phase inducing kinase. Schizosaccharomyces pombe Wee1 is a large protein, consisting of a C-terminal catalytic domain of approximately 350 amino acids preceded by a N-terminal domain of approximately 550 residues. The functional properties of the Wee1 N-terminal domain were investigated by expressing truncated forms of Wee1 in S. pombe. Both positive and negative regulatory domains were identified. Sequences important for Wee1 function were mapped to a central region (residues 363-408). This region is not required for kinase activity or nuclear localization, suggesting it may be involved in substrate recognition. The negative regulatory domain resides in the N-terminal third of Wee1, Wee1 constructs lacking this domain are more effective at delaying mitosis than wild-type Wee1. The negative regulatory domain contains clusters of potential Cdc2 phosphorylation sites. Investigations to monitor the abundance of Wee1 mRNA and protein during the cell cycle were also carried out.     

Overexpression of C-terminal Src kinase homologous kinase suppresses activation of Lyn tyrosine kinase required for VLA5-mediated Dami cell spreading

The Csk homologous kinase (Chk), which is co-expressed with C-terminal Src kinase (Csk) in hematopoietic cells, negatively regulates Src family kinases in vitro with selectivity toward Lyn but not c-Src in platelets. To explore the role of Src family kinases in hematopoietic cell adhesion, we overexpressed Chk in the megakaryocytic cell line Dami and established clones exhibiting a 10-fold increase in the amount of Chk. Overexpression of Chk was found to suppress VLA5 integrin-mediated cell spreading, but not cell attachment, throughout fibronectin (FN) stimulation. Deletion and point mutagenesis analyses of Chk showed that this suppression was dependent upon both the SH3 domain, which is responsible for membrane anchoring, and kinase activity. FN-induced cell spreading accompanied a sustained increase in Lyn activity with coincidental kinetics and the activation of Lyn was also suppressed by overexpression of Chk but not a Chk mutant lacking the SH3 domain. Expression of a truncated Lyn mutant lacking the kinase domain inhibited both cell spreading and Lyn activation upon stimulation with FN. These results suggest that sustained activation of Lyn, which is regulated by membrane-anchored Chk, plays a crucial role in VLA5-mediated cell spreading but not cell attachment to a FN substrate.     

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.     

Regulation of cell motility by mitogen-activated protein kinase

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.     

Receptor-operated calcium influx mediated by protein tyrosine kinase pathways

Calcium influx from the extracellular space elicited by activation of heterotrimeric G protein-coupled and heptahelical receptors plays a critical role in transmembrane signal transduction in a wide variety of cell systems. In nonexcitable cells, the precise voltage-independent mechanism by which calcium enters the cell remains unknown. Multiple mechanisms appear to be operating in different cell types (1-3): 1. G protein-operated calcium influx, 2. Second messenger-operated calcium influx, 3. Capacitative calcium influx, and 4. Phosphorylation of calcium channels. Receptor-operated calcium channels have a fundamental role in stimulus-secretion coupling in many different cells, but these channels remain to be purified and cloned. This review proposes that receptor-operated calcium influx is mediated by protein tyrosine kinase pathways. The function of protein tyrosine kinase pathways and their interactions with other receptor-operated calcium influx mechanisms are described.     

Regulation of neuronal survival by the serine-threonine protein kinase Akt

A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70(S6K)). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70(S6K) mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.     

Src family tyrosine kinase Lyn binds several proteins including paxillin in rat basophilic leukemia cells

Aggregation of the high affinity IgE receptors on rat basophilic leukemia (RBL-2H3) cells results in protein tyrosine phosphorylation although the receptor has no intrinsic enzymatic activity. The Src related protein tyrosine kinase p53/56lyn present in RBL-2H3 cells could play a role in this reaction. Here we have isolated the cDNA for rat Lyn and found it to be very homologous at the amino acid level to both the human and mouse proteins. A bacterially expressed maltose binding protein-Lyn (MBP-Lyn) fusion protein was already tyrosine phosphorylated and had tyrosine kinase activity. In a filter-binding assay, MBP-Lyn fusion protein (at 0.1 microM) specifically bound to several proteins of RBL-2H3 cells. In lysates of IgE receptor-activated cells, there was increased binding of MBP-Lyn to 65, 72, 78 and 110 kDa tyrosine phosphorylated proteins. The 72, 78 and 110 kDa tyrosine phosphorylated proteins were precipitated by a fusion protein containing the Lyn Src Homology 2 (SH2) domain. The 72 kDa Lyn binding protein was different from p72syk. Furthermore, paxillin, a cytoskeletal protein, was identified as one of the Lyn binding proteins. Thus Fc epsilon RI mediated signal transduction in RBL-2H3 cells may result from the interaction of p53/56lyn with paxillin, pp72, pp110 and other proteins.