Adaptive reprogramming of the breast cancer kinome.

Our understanding of cancer has grown considerably with recent advances in high-throughput genome and transcriptome sequencing, but techniques to comprehensively analyze protein activity are still in development. Methods to quantitatively measure the activation of signaling pathways within tumors at baseline and following therapeutic intervention will prove critical to the design of proper treatment regimens. Focusing on breast cancer, we present such a method to understand kinase signaling using multiplexed kinase inhibitor beads coupled with mass spectrometry (MIB/MS).

Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia.

Acute myeloid leukemia (AML) continues to be extremely difficult to treat successfully, and the unacceptably low overall survival rates mandate that we assess new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation in AML has been associated with poor prognosis and provides strategic targets for novel therapy development. We found that Mer receptor tyrosine kinase was over-expressed in a majority of pediatric (29/36, 80%) and adult (10/10, 100%) primary AML patient blasts at the time of diagnosis, and 100% of patient samples at the time of relapse. Mer was also found to be expressed in 12 of 14 AML cell lines (86%). In contrast, normal bone marrow myeloid precursors expressed little to no Mer. Following AML cell line stimulation with Gas6, a Mer ligand, we observed activation of prosurvival and proliferative signaling pathways, including phosphorylation of ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. To assess the phenotypic role of Mer in AML, two independent short-hairpin RNA (shRNA) constructs were used to decrease Mer expression in the AML cell lines Nomo-1 and Kasumi-1. Reduction of Mer protein levels significantly increased rates of myeloblast apoptosis two to threefold in response to serum starvation. Furthermore, myeloblasts with knocked-down Mer demonstrated decreased colony formation by 67-87%, relative to control cell lines (P<0.01). NOD-SCID-gamma mice transplanted with Nomo-1 myeloblasts with reduced levels of Mer had a significant prolongation in survival compared with mice transplanted with the parental or control cell lines (median survival 17 days in parental and control cell lines, versus 32-36 days in Mer knockdown cell lines, P<0.0001). These data suggest a role for Mer in acute myeloid leukemogenesis and indicate that targeted inhibition of Mer may be an effective therapeutic strategy in pediatric and adult AML.

Dasatinib inhibits site-specific tyrosine phosphorylation of androgen receptor by Ack1 and Src kinases.

Activation of androgen receptor (AR) may have a role in the development of castration-resistant prostate cancer. Two intracellular tyrosine kinases, Ack1 (activated cdc42-associated kinase) and Src, phosphorylate and enhance AR activity and promote prostate xenograft tumor growth in castrated animals. However, the upstream signals that activate these kinases and lead to AR activation are incompletely characterized. In this study, we investigated AR phosphorylation in response to non-androgen ligand stimulation using phospho-specific antibodies. Treatment of LNCaP and LAPC-4 cells with epidermal growth factor (EGF), heregulin, Gas6 (ligand binding to the Mer receptor tyrosine kinase and activating Ack1 downstream), interleukin (IL)-6 or bombesin stimulated cell proliferation in the absence of androgen. Treatment of LNCaP and LAPC-4 cells with EGF, heregulin or Gas6 induced AR phosphorylation at Tyr-267, whereas IL-6 or bombesin treatment did not. AR phosphorylation at Tyr-534 was induced by treatment with EGF, IL-6 or bombesin, but not by heregulin or Gas6. Small interfering RNA-mediated knockdown of Ack1 or Src showed that Ack1 mediates heregulin- and Gas6-induced AR Tyr-267 phosphorylation, whereas Src mediates Tyr-534 phosphorylation induced by EGF, IL-6 and bombesin. Dasatinib, a Src inhibitor, blocked EGF-induced Tyr-534 phosphorylation. In addition, we showed that dasatinib also inhibited Ack1 kinase. Dasatinib inhibited heregulin-induced Ack1 kinase activity and AR Tyr-267 phosphorylation. In addition, dasatinib inhibited heregulin-induced AR-dependent reporter activity. Dasatinib also inhibited heregulin-induced expression of endogenous AR target genes. Dasatinib inhibited Ack1-dependent colony formation and prostate xenograft tumor growth in castrated mice. Interestingly, Ack1 or Src knockdown or dasatinib did not inhibit EGF-induced AR Tyr-267 phosphorylation or EGF-stimulated AR activity, suggesting the existence of an additional tyrosine kinase that phosphorylates AR at Tyr-267. These data suggest that specific tyrosine kinases phosphorylate AR at distinct sites and that dasatinib may exert antitumor activity in prostate cancer through inhibition of Ack1.

ErbB-2 via PYK2 upregulates the adhesive ability of androgen receptor-positive human prostate cancer cells.

Aberrant regulation in the adhesive ability of cancer cells is closely associated with their metastatic activity. In this study, we examine the role of ErbB-2 in regulating the adhesive ability of androgen receptor (AR)-positive human prostate cancer (PCa) cells, the major cell population of PCa. Utilizing different LNCaP and MDA PCa2b cells as model systems, we found that ErbB-2 activity was correlated with PYK2 activity and adhesive ability in those cells. Increased ErbB-2 expression or activity in LNCaP C-33 cells enhanced PYK2 activation and cell adhesion, while the high PYK2 activity and the rapid adhesion of LNCaP C-81 cells were decreased by diminishing ErbB-2 expression or activity. Knockdown studies revealed the predominant role of ErbB-2 in regulating LNCaP C-81 cell adhesion. Coimmunoprecipitation showed that C-81 cells had increased interaction between ErbB-2 and PYK2. Elevated ErbB-2 activity in LNCaP cells correlated with increased ERK/MAPK activity and enhanced adhesive ability, which were abolished by the expression of K457A-PYK2 mutant or the treatment of PD98059, a MEK inhibitor. In summary, our data suggested that ErbB-2, via PYK2-ERK/MAPK, upregulates the adhesive ability of AR-positive human PCa cells.

Her4 mediates ligand-dependent antiproliferative and differentiation responses in human breast cancer cells.

The function of the epidermal growth factor receptor (EGFR) family member HER4 remains unclear because its activating ligand, heregulin, results in either proliferation or differentiation. This variable response may stem from the range of signals generated by HER4 homodimers versus heterodimeric complexes with other EGFR family members. The ratio of homo- and heterodimeric complexes may be influenced both by a cell's EGFR family member expression profile and by the ligand or even ligand isoform used. To define the role of HER4 in mediating antiproliferative and differentiation responses, human breast cancer cell lines were screened for responses to heregulin. Only cells that expressed HER4 exhibited heregulin-dependent antiproliferative responses. In-depth studies of one line, SUM44, demonstrated that the antiproliferative and differentiation responses correlated with HER4 activation and were abolished by stable expression of a kinase-inactive HER4. HB-EGF, a HER4-specific ligand in this EGFR-negative cell line, also induced an antiproliferative response. Moreover, introduction and stable expression of HER4 in HER4-negative SUM102 cells resulted in the acquisition of a heregulin-dependent antiproliferative response, associated with increases in markers of differentiation. The role of HER2 in these heregulin-dependent responses was examined through elimination of cell surface HER2 signaling by stable expression of a single-chain anti-HER2 antibody that sequestered HER2 in the endoplasmic reticulum. In the cell lines with either endogenously (SUM44) or exogenously (SUM102) expressed HER4, elimination of HER2 did not alter HER4-dependent decreases in cell growth. These results suggest that HER4 is both necessary and sufficient to trigger an antiproliferative response in human breast cancer cells.

Phagocytosis and clearance of apoptotic cells is mediated by MER.

Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

Inhibition of the calcium-dependent tyrosine kinase (CADTK) blocks monocyte spreading and motility.

Freshly isolated peripheral blood monocytes lack focal adhesion kinase (p125(FAK)) but activate a second member of this kinase family, calcium-dependent tyrosine kinase (CADTK; also known as Pyk2/CAKbeta/RAFTK/FAK2), upon adhesion or stimulation with chemokines. To study the role of CADTK in monocyte adherence and motility, we performed immunocytochemical localization that showed CADTK at the leading edge and ruffling lamellipodial structures in freshly isolated, adhered human monocytes. We next introduced CADTK/CAKbeta-related non-kinase (CRNK), the C-terminal noncatalytic domain of CADTK, into monocytes by electroporation and showed that it inhibited CADTK autophosphorylation. Introduction of the fusion protein glutathione S-transferase (GST)-CRNK also reduced (i) cell spreading, as reflected in a reduced cell area 30 min after adhesion, (ii) adhesion-induced phosphotyrosine increases and redistribution into lamellipodia, and (iii) adhesion-induced extracellular signal-regulated protein kinase (ERK) activation. In control experiments, introduction of GST or GST-C3 transferase (an inhibitor of RhoA GTPase activity) by electroporation did not affect these parameters. Monocytes adhered in the presence of autologous serum were highly motile even after introduction of GST (83% motile cells). However, only 26% of monocytes with introduced GST-CRNK were motile. In contrast, GST-CRNK-treated monocytes were fully capable of phagocytosis and adhesion-induced cytokine gene induction, suggesting that CADTK is not involved in these cellular activities and that GST-CRNK introduction does not inhibit global monocyte functions. These results suggest that CADTK is crucial for the in vitro monocyte cytoskeletal reorganization necessary for cell motility and is likely to be required in vivo for recruitment to sites of inflammation.

Regulation of carbamoyl phosphate synthetase by MAP kinase.

The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by carbamoyl phosphate synthetase (CPS II), part of the multifunctional enzyme CAD. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo, CAD lost its feedback inhibition (which is dependent on uridine triphosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyrophosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triphosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides.

Tyro-3 family receptors are essential regulators of mammalian spermatogenesis.

We have generated and analysed null mutations in the mouse genes encoding three structurally related receptors with tyrosine kinase activity: Tyro 3, Axl, and Mer. Mice lacking any single receptor, or any combination of two receptors, are viable and fertile, but male animals that lack all three receptors produce no mature sperm, owing to the progressive death of differentiating germ cells. This degenerative phenotype appears to result from a failure of the tropic support that is normally provided by Sertoli cells of the seminiferous tubules, whose function depends on testosterone and additional factors produced by Leydig cells. Tyro 3, Axl and Mer are all normally expressed by Sertoli cells during postnatal development, whereas their ligands, Gas6 and protein S, are produced by Leydig cells before sexual maturity, and by both Leydig and Sertoli cells thereafter. Here we show that the concerted activation of Tyro 3, Axl and Mer in Sertoli cells is critical to the role that these cells play as nurturers of developing germ cells. Additional observations indicate that these receptors may also be essential for the tropic maintenance of diverse cell types in the mature nervous, immune and reproductive systems.

A novel receptor tyrosine kinase, Mer, inhibits TNF-alpha production and lipopolysaccharide-induced endotoxic shock.

The regulation of monocyte function and the inhibition of TNF-alpha production during bacterial sepsis are critical in attenuating adverse host responses to endotoxemia. To study the function of a novel receptor tyrosine kinase, mer, that is expressed in monocytes, we generated mice (merkd) that lack the signaling tyrosine kinase domain. Upon LPS challenge, merkd animals died of endotoxic shock (15/17, 88.2%), whereas control wild-type mice survived (1/15, 6.7% died). Susceptible merkd mice exhibited edema, leukocyte infiltration, and signs of endotoxic shock that correlated with higher levels of TNF-alpha found in the serum of merkd mice as compared with wild-type control animals. Death due to LPS-induced endotoxic shock in merkd mice was blocked by administration of anti-TNF-alpha Ab, suggesting that overproduction of this cytokine was principally responsible for the heightened suseptibility. The increase in TNF-alpha production appeared to be the result of a substantial increase in the LPS-dependent activation of NF-kappa B nuclear translocation resulting in greater TNF-alpha production by macrophages from merkd mice. Thus, Mer receptor tyrosine kinase signaling participates in a novel inhibitory pathway in macrophages important for regulating TNF-alpha secretion and attenuating endotoxic shock.

Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase.

The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKbeta/FAK2, is a cytoskeleton-associated tyrosine kinase. We compared CADTK regulation with that of the highly homologous focal adhesion tyrosine kinase (FAK). First, we generated site-specific CADTK mutants. Mutation of Tyr402 eliminated autophosphorylation and significantly decreased kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation site predicted to bind Grb2, had little effect on CADTK regulation. Src family tyrosine kinases resulted in CADTK tyrosine phosphorylation even when co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK tyrosine-phosphorylated FAK when both were transiently expressed, but FAK did not phosphorylate CADTK. Biochemical experiments confirmed direct CADTK phosphorylation of FAK. This phosphorylation utilized tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Last, expression of the CADTK carboxyl terminus (CRNK) abolished CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibited both FAK and CADTK autophosphorylation, suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling.

Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) produces intracellular calcium and protein kinase C (PKC) signals and stimulates ERK and JNK activity. JNK activation appears to be mediated by a calcium-dependent tyrosine kinase (CADTK). To define the ERK pathway, we established GN4 cells expressing an inhibitory Ras(N17). Induction of Ras(N17) blocked EGF- but not Ang II- or phorbol ester (TPA)-dependent ERK activation. In control cells, Ang II and TPA produced minimal increases in Ras-GTP level and Raf kinase activity. PKC depletion by chronic TPA exposure abolished TPA-dependent ERK activation but failed to diminish the effect of Ang II. In PKC-depleted cells, Ang II increased Ras-GTP level and activated Raf and ERK in a Ras-dependent manner. In PKC depleted cells, Ang II stimulated Shc and Cbl tyrosine phosphorylation, suggesting that without PKC, Ang II activates another tyrosine kinase. PKC-depletion did not alter Ang II-dependent tyrosine phosphorylation or activity of p125(FAK), CADTK, Fyn or Src, but PKC depletion or incubation with GF109203X resulted in Ang II-dependent EGF receptor tyrosine phosphorylation. In PKC-depleted cells, EGF receptor-specific tyrosine kinase inhibitors blocked Ang II-dependent EGF receptor and Cbl tyrosine phosphorylation, and ERK activation. In summary, Ang II can activate ERK via two pathways; the latent EGF receptor, Ras-dependent pathway is equipotent to the Ras-independent pathway, but is masked by PKC action. The prominence of this G-protein coupled receptor to EGF receptor pathway may vary between cell types depending upon modifiers such as PKC.

A calcium-dependent tyrosine kinase splice variant in human monocytes. Activation by a two-stage process involving adherence and a subsequent intracellular signal.

Freshly isolated human monocytes do not express p125(FAK) but upon adherence to substrata activate the highly related calcium-dependent tyrosine kinase (CADTK), also known as Pyk2, CAKbeta, RAFTK, and FAK2. The monocyte CADTK was 5 kDa smaller than protein from epithelial cells; isolation and sequencing of the monocyte CADTK cDNA revealed a predicted 42-amino acid deletion between the two proline-rich domains of the enzyme. The nucleic acid sequence suggests that the deletion is caused by alternative RNA splicing. This species was also found in T and B lymphocytes and appears to be the predominant form of cytoskeletal associated tyrosine kinase in non-neoplastic, circulating, hematopoietic cells. CADTK was not activated when monocytes maintained in suspension were treated with agents that produce an intracellular calcium (thapsigargin) or protein kinase C (phorbol 12-myristate 13-acetate) signal including a chemokine, RANTES, that binds to the HIV co-receptor, CCK5. In contrast, monocyte adherence to tissue culture plastic-stimulated CADTK tyrosine phosphorylation, a process that was enhanced by thapsigargin, phorbol 12-myristate 13-acetate, and RANTES but that was completely blocked by preincubation with cytochalasin D. When compared with plastic, adherence to fibronectin- or collagen-coated surfaces produced only minimal CADTK activation but permitted significant stimulation by added thapsigargin. These data suggest that in a cell type that lacks p125(FAK), CADTK plays an early role in post-adherence signaling. Its activation involves two stages, cytoskeletal engagement, which is permissive, and co-stimulatory signals (calcium or protein kinase C) generated by extensive cell surface engagement, agonists, or inflammatory chemokines.

Regulation of a calcium-dependent tyrosine kinase in vascular smooth muscle cells by angiotensin II and platelet-derived growth factor. Dependence on calcium and the actin cytoskeleton.

A novel, p125FAK homologue, CADTK, has been detected in neural, epithelial, or hematopoietic cells but not in fibroblasts. We now demonstrate CADTK expression in a mesenchymal cell, rat aortic smooth muscle cells (RSMC). Angiotensin II (Ang II) or platelet-derived growth factor (PDGF-BB and PDGF-AA) markedly stimulated CADTK tyrosine phosphorylation in RSMC but did not affect p125FAK phosphorylation. The PDGF-depedent CADTK tyrosine phosphorylation was slower and more prolonged than that of Ang II, correlating well with the differential effects of these agonists on cytosolic calcium ([Ca2+]i) signaling. An intracellular calcium chelator inhibited both the rapid and sustained activation of CADTK by Ang II and PDGF. Extracellular calcium chelation inhibited the PDGF-stimulated increase in CADTK tyrosine phosphorylation as well as the sustained (but not the early) activation by Ang II. In contrast, p125FAK tyrosine phosphorylation was maximal in quiescent, adherent RSMC and was not affected by incubation with EGTA. Depletion of protein kinase C activity partially inhibited both the Ang II- and PDGF-induced CADTK tyrosine phosphorylation. Additional results confirm a relation between CADTK and the cytoskeleton. First, the tyrosine phosphorylation of paxilin correlated with activation of CADTK; this increase was inhibited by EGTA. Second, cytochalasin D blocked the PDGF- or Ang II-stimulated tyrosine phosphorylation of CADTK, suggesting a role for the cytoskeleton in agonist-dependent CADTK activation. Third, immunofluorescence analysis of CADTK localization demonstrated actin-like cytoskeleton staining extending into focal contacts. These results suggest that in mesenchymal cells, CADTK is localized to and activated by an actin cytoskeleton-dependent mechanism; a mechanism that is regulated in a calcium and protein kinase C-dependent manner independently of p125FAK.

Osmotic shock stimulates GLUT4 translocation in 3T3L1 adipocytes by a novel tyrosine kinase pathway.

Similar to insulin, osmotic shock of 3T3L1 adipocytes stimulated an increase in glucose transport activity and translocation of GLUT4 protein from intracellularly localized vesicles to the plasma membrane. The docking/fusion of GLUT4 vesicles with the plasma membrane appeared to utilize a similar mechanism, since expression of a dominant interfering mutant of syntaxin-4 prevented both insulin- and osmotic shock-induced GLUT4 translocation. However, although the insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, activation by osmotic shock was wortmannin-insensitive. Furthermore, insulin stimulated the phosphorylation and activation of the Akt kinase, whereas osmotic shock was completely without effect. Surprisingly, treatment of cells with the tyrosine kinase inhibitor, genistein, or microinjection of phosphotyrosine antibody prevented both the insulin- and osmotic shock-stimulated translocation of GLUT4. In addition, osmotic shock induced the tyrosine phosphorylation of several discrete proteins including Cbl, p130(cas), and the recently identified soluble tyrosine kinase, calcium-dependent tyrosine kinase (CADTK). In contrast, insulin had no effect on CADTK but stimulated the tyrosine phosphorylation of Cbl and the tyrosine dephosphorylation of pp125(FAK) and p130(cas). These data demonstrate that the osmotic shock stimulation of GLUT4 translocation in adipocytes occurs through a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.

Protein kinase C and protein kinase A inhibit calcium-dependent but not stress-dependent c-Jun N-terminal kinase activation in rat liver epithelial cells.

In rat liver epithelial cells (GN4), angiotensin II (Ang II) and thapsigargin stimulate a novel calcium-dependent tyrosine kinase (CADTK) also known as PYK2, CAKbeta, or RAFTK. Activation of CADTK by a thapsigargin-dependent increase in intracellular calcium failed to stimulate the extracellular signal-regulated protein kinase pathway but was well correlated with a 30-50-fold activation of c-Jun N-terminal kinase (JNK). In contrast, Ang II, which increased both protein kinase C (PKC) activity and intracellular calcium, stimulated extracellular signal-regulated protein kinase but produced a smaller, less sustained, JNK activation than thapsigargin. 12-O-Tetradecanoylphorbol 13-acetate (TPA), which slowly activated CADTK, did not stimulate JNK. These findings suggest either that CADTK is not involved in JNK activation or PKC activation inhibits the CADTK to JNK pathway. A 1-min TPA pretreatment of GN4 cells inhibited thapsigargin-dependent JNK activation by 80-90%. In contrast, TPA did not inhibit the >50-fold JNK activation effected by anisomycin or UV. The consequence of PKC-dependent JNK inhibition was reflected in c-Jun and c-Fos mRNA induction following treatment with thapsigargin and Ang II. Thapsigargin, which only minimally induced c-Fos, produced a much greater and more prolonged c-Jun response than Ang II. Elevation of another intracellular second messenger, cAMP, for 5-15 min also inhibited calcium-dependent JNK activation by approximately 80-90% but likewise had no effect on the stress-dependent JNK pathway. In summary, two pathways stimulate JNK in cells expressing CADTK, a calcium-dependent pathway modifiable by PKC and cAMP-dependent protein kinase and a stress-activated pathway independent of CADTK, PKC, and cAMP-dependent protein kinase; the inhibition by PKC can ultimately alter gene expression initiated by a calcium signal.

Paxillin is tyrosine-phosphorylated by and preferentially associates with the calcium-dependent tyrosine kinase in rat liver epithelial cells.

We and others have recently cloned a non-receptor, calcium-dependent tyrosine kinase (CADTK; also known as PYK2, CAKbeta, and RAFTK) that shares both overall domain structure and 45% amino acid identity with p125(FAK). We have studied the signaling, activation, and potential function of these related enzymes in GN4 rat liver epithelial cells that express CADTK and p125(FAK) at roughly similar levels. p125(FAK) is nearly fully tyrosine-phosphorylated in resting GN4 cells. In contrast, while CADTK is not tyrosine-autophosphorylated in untreated cells, angiotensin II increases CADTK Tyr(P) by 5-10-fold. With regard to signaling, CADTK activation is correlated with stimulation of c-Jun N-terminal kinase and p70(S6K) pathways but not with the stimulation of mitogen-activated protein kinase or p90(RSK). In this report we assessed the contribution of CADTK and p125(FAK) to tyrosine phosphorylation of focal contact proteins. In adherent GN4 cells, the constitutive activity of p125(FAK) was correlated with basal paxillin, tensin, and p130(CAS) tyrosine phosphorylation. A rapid increase in the tyrosine phosphorylation of each protein was detected after treatment with angiotensin II or other agonists that stimulate CADTK; the prolonged 3-4-fold increase in paxillin tyrosine phosphorylation was the most substantial change. In the WB cell line that expresses 3-fold less CADTK than GN4 cell line agonist-dependent paxillin tyrosine phosphorylation is similarly reduced. Immunoprecipitation of CADTK from GN4 cells revealed CADTK. paxillin complexes that persisted in 500 mM NaCl but not in 0.1% SDS cell lysis buffer. The complexes were largely independent of the tyrosine phosphorylation state of either protein. Surprisingly, we did not detect p125(FAK).paxillin complexes in immunoprecipitates using either of two p125(FAK) antibodies. When CADTK and p125(FAK) were transiently overexpressed in 293(T) cells, both enzymes associated with paxillin, but the avidity of CADTK appeared to be greater. In addition, in transfected 293(T) cells, complexes between CADTK and another potential substrate, p130(CAS), were detected. In summary, in GN4 rat liver epithelial cells stimulation of CADTK was highly correlated with paxillin tyrosine phosphorylation; in addition, CADTK but not p125(FAK) was complexed to paxillin at detectable levels. This suggests that agonist-dependent cytoskeletal changes in epithelial cells might proceed, in part, by CADTK-dependent mechanisms.

An intracellular calcium signal activates p70 but not p90 ribosomal S6 kinase in liver epithelial cells.

In the rat liver epithelial cell lines GN4 and WB, angiotensin II (Ang II) activates the Gq class of regulatory G-proteins, increasing intracellular calcium, protein kinase C activity, and protein tyrosine phosphorylation. We compared the ability of Ang II and other compounds that increase intracellular calcium (i.e. the calcium ionophore A23187 and thapsigargin) or protein kinase C activity (the phorbol ester 12-O-tetradecanoylphorbol-13-acetate) to activate p70 ribosomal S6 kinase (p70(S6K)) and p90 ribosomal S6 kinase (p90(RSK)). In GN4 cells, increasing intracellular calcium stimulated p70(S6K) activity in a rapamycin- and wortmannin- sensitive manner, but did not affect p90(RSK) activity. In contrast, 12-O-tetradecanoylphorbol-13-acetate strongly activated p90(RSK) but only weakly stimulated p70(S6K). The ability of calcium to activate p70(S6K) was confirmed by blocking the A23187-dependent activation through chelation of extracellular calcium with EGTA; the effect of thapsigargin was inhibited by the cell permeant chelator bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Similarly, BAPTA-AM prevented the activation of p70(S6K) by Ang II, suggesting that this signal was largely calcium-dependent. In contrast, the Ang II-dependent activation of mitogen-activated protein kinase and p90(RSK) was not inhibited but was enhanced by BAPTA-AM. These results show that in GN4 cells, Ang II selectively activates p70(S6K) through effects on calcium, p90(RSK) through effects on protein kinase C. The activation of p70(S6K) by calcium stimuli or Ang II was independent of calmodulin but correlated well with the activation of the recently identified, nonreceptor calcium-dependent tyrosine kinase (CADTK)/PYK-2. Both calcium- and Ang II-dependent activation of p70(S6K) were attenuated by the tyrosine kinase inhibitor genistein, and activation of p70(S6K) was higher in GN4 than WB cells, correlating with the increased expression and activation of CADTK/PYK-2 in GN4 cells. In summary, these results demonstrate that intracellular calcium selectively activates p70(S6K) in GN4 cells, consistent with increased CADTK/PYK-2 signaling in these cells.

Activation of a novel calcium-dependent protein-tyrosine kinase. Correlation with c-Jun N-terminal kinase but not mitogen-activated protein kinase activation.

Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase Cbeta, initially increasing intracellular calcium and activating protein kinase C. In the WB and GN4 rat liver epithelial cell lines, agonist-induced calcium signals also stimulate tyrosine phosphorylation and subsequently increase the activity of c-Jun N-terminal kinase (JNK). We have now purified the major calcium-dependent tyrosine kinase (CADTK), and by peptide and nucleic acid sequencing identified it as a rat homologue of human PYK2. CADTK/PYK2 is most closely related to p125(FAK) and both enzymes are expressed in WB and GN4 cells. Angiotensin II, which only slightly increases p125(FAK) tyrosine phosphorylation in GN4 cells, substantially increased CADTK tyrosine autophosphorylation and kinase activity. Agonists for other G protein-coupled receptors (e.g. LPA), or those increasing intracellular calcium (thapsigargin), also stimulated CADTK. In comparing the two rat liver cell lines, GN4 cells exhibited approximately 5-fold greater angiotensin II- and thapsigargin-dependent CADTK activation than WB cells. Although maximal JNK activation by stress-dependent pathways (e.g. UV and anisomycin) was equivalent in the two cell lines, calcium-dependent JNK activation was 5-fold greater in GN4, correlating with CADTK activation. In contrast to JNK, the thapsigargin-dependent calcium signal did not activate mitogen-activated protein kinase and Ang II-dependent mitogen-activated protein kinase activation was not correlated with CADTK activation. Finally, while some stress-dependent activators of the JNK pathway (NaCl and sorbitol) stimulated CADTK, others (anisomycin, UV, and TNFalpha) did not. In summary, cells expressing CADTK/PYK2 appear to have two alternative JNK activation pathways: one stress-activated and the other calcium-dependent.