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.

Calcium-dependent increase in tyrosine kinase activity stimulated by angiotensin II.

The cellular effects of numerous hormones and neurotransmitters, including the vasoactive agents angiotensin II (AngII) and [Arg8]vasopressin, are mediated in part by protein-serine threonine kinases activated by increase of cytosolic Ca2+ concentration. In this study, we have tested the ability of Ca(2+)-mobilizing agents to activate cellular tyrosine kinases. Treatment of intact GN4 liver epithelial cells with AngII rapidly (less than or equal to 15 sec) increased tyrosine kinase activity measured either in unfractionated cell lysates or in anti-phosphotyrosine immune complexes from detergent-solubilized cells. Increased phosphorylation of the exogenous substrate poly(Glu80Tyr20) (3- to 4-fold over control) by immunoprecipitated kinases closely paralleled the time- and dose-dependence of the appearance of tyrosine phosphoproteins in intact cells. This effect of AngII was mimicked by thapsigargin, a Ca(2+)-elevating tumor promoter. The ability of AngII, but not epidermal growth factor, to increase tyrosine kinase activity was blocked in cells loaded with the Ca2+ chelator bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. Dephosphorylation of immunoprecipitated proteins by tyrosine phosphatase treatment was accompanied by a 60-70% loss in in vitro kinase activity, suggesting that the AngII-sensitive kinase(s) are activated by phosphorylation in intact cells. These findings demonstrate a link between two widely occurring signaling pathways, the tyrosine kinases and the Ca2+ second-messenger system, and suggest the possible involvement of Ca(2+)-activated tyrosine kinases in the endocrine actions of AngII and [Arg8]vasopressin.

Angiotensin II stimulates protein-tyrosine phosphorylation in a calcium-dependent manner.

Cellular responses to epidermal growth factor (EGF) are dependent on the tyrosine-specific protein kinase activity of the cell-surface EGF receptor. Previous studies using WB rat liver epithelial cells have detected at least 10 proteins whose phosphotyrosine (P-Tyr) content is increased by EGF. In this study, we have examined alternate modes of activating tyrosine phosphorylation. Treatment of WB cells with hormones linked to Ca2+ mobilization and protein kinase C (PKC) activation, including angiotensin II, [Arg8]vasopressin, or epinephrine, stimulated rapid (less than or equal to 15-s) and transient increases in the P-Tyr content of several proteins (p120/125, p75/78, and p66). These proteins, detected by anti-P-Tyr immunoblotting, were similar in molecular weight to a subset of EGF-sensitive P-Tyr-containing proteins (P-Tyr-proteins). The increased P-Tyr content was confirmed by [32P]phosphoamino acid analysis of proteins recovered by anti-P-Tyr immunoprecipitation. Elevating intracellular [Ca2+] with the ionophore A23187 or ionomycin or with the tumor promoter thapsigargin mimicked the effects of hormones on tyrosine phosphorylation, whereas treatment with a PKC-activating phorbol ester did not. In addition, responses to angiotensin II were not diminished in PKC-depleted cells. Ca2+ mobilization, measured by fura-2 fluorescence, was coincident with the increase in tyrosine phosphorylation in response to angiotensin II or thapsigargin. Loading cells with the intracellular Ca2+ chelator bis-(o-aminophenoxy)ethane-N ,N ,N' , N'-tetraacetic acid (BAPTA) inhibited the appearance of all P-Tyr-proteins in response to angiotensin II, thapsigargin, or ionophores, as well as two EGF-stimulated P-Tyr-proteins. The majority of EGF-stimulated P-Tyr-proteins were not affected by BAPTA. These studies indicate that angiotensin II can alter protein-tyrosine phosphorylation in a manner that is secondary to, and apparently dependent on, Ca2+ mobilization. Thus, ligands such as EGF and angiotensin II, which act through distinct types of receptors, may activate secondary pathways involving tyrosine phosphorylation. These results also raise the possibility that certain growth-promoting effects of Ca2+ -mobilizing agents such as angiotensin II may be mediated via tyrosine phosphorylation.

Vanadate stimulates tyrosine phosphorylation of two proteins in Raji human lymphoblastoid cell membranes.

A membrane fraction from Raji human lymphoblastoid cells exhibited tyrosine-specific kinase activity. Vanadate increased tyrosine phosphorylation up to 5-fold; serine and threonine phosphorylation were unchanged. The stimulation was detectable within 15 s at 0 degrees C and at concentrations of vanadate (0.3 and 1.0 microM) present in normal tissues and blood. The tyrosine phosphorylation of two substrates, M1 61 000 and 55 000, was dependent upon vanadate and incorporation into these substrates represented the majority of the vanadate-sensitive tyrosine phosphorylation.

DMSO increases tyrosine residue phosphorylation in membranes from murine erythroleukemia cells.

Phosphorylation of membranes from murine erythroleukemia cells was performed in the presence and absence of the polar solvent dimethyl sulfoxide. Quantitation of the phosphoamino acid content revealed that DMSO stimulated phosphotyrosine accumulation by three-fold; serine and threonine phosphorylation decreased significantly. We had previously shown that DMSO stimulated tyrosine residue phosphorylation of the hepatic epidermal growth factor receptor. EGF had little effect in MEL membranes; therefore, DMSO results in accumulation of phosphotyrosine in cell membranes that do not exhibit significant EGF-dependent phosphorylation.

Structural and functional regression of polycystic ovaries by danazol.

The effects of danazol on ovarian steroidogenesis and follicular size was studied in androgen-sterilized rats bearing polycystic ovaries. Daily treatment with 4 or 20 mg of the drug caused a reduction in ovarian weight and regression of cystic follicles. Serum levels of luteinizing hormone (LH), testosterone, and estrogen were significantly decreased. The persistent estrus smears were changed to diestrus, indicating inhibition of estrogenic activity.