Focal adhesion kinase tyrosine-861 is a major site of phosphorylation by Src.

Focal adhesion kinase (FAK) participates in signaling events induced by diverse stimuli including integrin engagement, oncogenic transformation and mitogenic neuropeptides. FAK's signaling function is regulated by tyrosine phosphorylation. The major autophosphorylation site is tyrosine-397, which interacts with the Src homology 2 (SH2) domain of Src-family kinases including Src and Fyn. Full activation of FAK appears to require additional phosphorylation by the associated Src-family kinases. Previously identified Src sites include catalytic domain tyrosines-576 and -577, important for maximal FAK kinase activity, and tyrosine-925, which permits an SH2-mediated association with Grb2. A full understanding of FAK-mediated signaling events will require the identification of all sites of tyrosine phosphorylation. Here we report that tyrosine-861 is the major Src site in the carboxyl-terminal domain of FAK. Phosphotyrosine-861 may function in additional interactions between FAK and SH2-containing proteins.

Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases.

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII--a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.

Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin.

A homology-based cDNA cloning approach was used to identify a widely expressed protein-tyrosine kinase designated as "focal adhesion kinase" (FadK). The entire mouse FadK amino acid sequence was deduced from cDNA clones, revealing a large (119-kDa) non-membrane-spanning protein-tyrosine kinase that lacks Src-homology SH2 and SH3 domains. Immunostaining of BALB/c 3T3 fibroblasts revealed that FadK is concentrated in focal adhesions. FadK is phosphorylated on tyrosine in growing cultures of BALB/c 3T3 cells but contains little or no phosphotyrosine in cells detached by trypsinization. The tyrosine-phosphorylated state is regained within minutes when the cells are replated onto fibronectin. Activation of FadK may be an important early step in intracellular signal transduction pathways triggered in response to cell interactions with the extracellular matrix.

Molecular cloning and enzymatic analysis of the rat homolog of "PhK-gamma T," an isoform of phosphorylase kinase catalytic subunit.

Messenger RNA encoding a protein kinase closely related to the catalytic subunit of skeletal muscle phosphorylase kinase has previously been isolated from a human HeLa cell cDNA library, and cross-species Northern hybridization analysis has shown that the rat homolog of this transcript is abundant in the adult testis (Hanks, S.K. (1989) Mol. Endocrinol. 3, 110-116). We now propose that the protein encoded by this transcript be designated as "PhK-gamma T." In this article, the primary structure of the rat homolog of PhK-gamma T is described, as deduced from nucleotide sequences of cDNA and genomic clones. RNase protection analysis reveals that PhK-gamma T transcripts are actually present in a wide variety of adult rat tissues, but at levels 20-100-fold less than what is observed in the testis. In the testis, transcription of the PhK-gamma T gene is initiated at multiple sites as shown by RNase protection and primer extension. Enzymatic activity of PhK-gamma T was demonstrated using renatured bacterially expressed protein. In the presence of Ca2+/calmodulin, PhK-gamma T is able to efficiently phosphorylate glycogen phosphorylase and convert it from an inactive to an active form. We conclude that PhK-gamma T represents a true isoform of phosphorylase kinase catalytic subunit.

Phosphorylation of calcineurin: effect on calmodulin binding.

The effect of phosphorylation of calcineurin on calmodulin (CaM) binding was examined using a synthetic peptide which contains the CaM-binding domain and the serine phosphorylation site. The peptide, corresponding to residues 391-414 of brain calcineurin A subunit, was rapidly phosphorylated by protein kinase C and Ca2+/CaM-dependent protein kinase II but not by cAMP-dependent protein kinase. Phosphorylation of peptide 391-414 did not significantly alter the binding of CaM when compared to the non-phosphorylated peptide.

Bicarbonate absorption stimulates active calcium absorption in the rat proximal tubule.

To evaluate the effect of luminal bicarbonate on calcium reabsorption, rat proximal tubules were perfused in vivo. Perfusion solution contained mannitol to reduce water flux to zero. Total Ca concentration was measured by atomic absorption spectrometry, Ca ion concentration in the tubule lumen (CaL2+) and the peritubular capillary (CaP2+), and luminal pH (pHL) with ion-selective microelectrodes and transepithelial voltage (VTE) with conventional microelectrodes. When tubules were perfused with buffer-free Cl-containing solution, net Ca absorption (JCa) averaged 3.33 pmol/min. Even though VTE was 1.64 mV lumen-positive, CaL2+, 1.05 mM, did not fall below the concentration in the capillary blood, 1.07 mM. When 27 mM of Cl was replaced with HCO3, there was luminal fluid acidification. Despite a decrease in VTE and CaL2+, JCa increased to 7.13 pmol/min, indicating that the enhanced JCa could not be accounted for by the reduced electrochemical gradient, delta CCa. When acetazolamide or an analogue of amiloride was added to the HCO3 solution, JCa was not different from the buffer-free solution, suggesting that HCO3-stimulated JCa may be linked to acidification. To further test this hypothesis, we used 27 mM Hepes as the luminal buffer. With Hepes there was luminal fluid acidification and JCa was not different from the buffer-free solution but delta CCa was significantly reduced, indicating enhanced active calcium transport. We conclude from the results of the present study that HCO3 stimulates active Ca absorption, a process that may be linked to acidification-mediated HCO3 absorption.

Recombinant murine interferon-gamma-induced differentiation of pre-B lymphocytes is associated with Na+/H+ exchange-dependent and -independent cytoplasmic alkalinization.

We have previously shown that in a murine pre-B lymphocyte cell line, 70Z/3, interleukin-1-induced IgM expression (differentiation) was associated with Na+/H+-mediated cytoplasmic alkalinization. Because interferon-gamma also induces 70Z/3 differentiation, in this study we examined the effects of recombinant murine interferon-gamma (rIFN-gamma) on cell pH. We found that rIFN-gamma (50 units/ml) induced a sustained increase in cell pH, averaging 0.09 pH units above the base line at 30 min and 0.08 at 4 h. Because rIFN-gamma also induced increases in cell Na+ concentration, the data suggests stimulation of Na+/H+ exchange across the cell membrane. Amiloride inhibited the rIFN-gamma-mediated pH rise by only 31% at 30 min, but at 4 h the inhibition was more complete (89%). In contrast to pHi, amiloride totally blocked the Na+ rise at both 30 min and 4 h. This indicates that at the earlier time point the pH rise had a Na+/H+ exchange-dependent and -independent component while at the later time most of the pH rise was due to the Na+/H+ exchanger. The presence of a Na+ independent amiloride-insensitive component was further confirmed by the 0.05 pH rise induced by rIFN-gamma in Na+-free media. Failure to block the Na+-independent rIFN-gamma-mediated pHi rise by anion exchange inhibitors or removal of Ca2+ indicate that this component was not mediated by Cl-/OH- or Ca2+/H+ exchange. The nature of the Na+-independent cell alkalinization process or its role in rIFN-gamma-mediated 70Z/3 differentiation remains to be determined. Because amiloride did not have an effect on rIFN-gamma-induced surface Ig expression or the rIFN-gamma-mediated increase in new kappa light chain-specific mRNA the results indicate that rIFN-gamma-stimulated Na+/H+ is not required for 70Z/3 differentiation.

A microelectrometric titration method for measurement of total intracellular Cl- concentration.

A microelectrometric titration method is described to measure picomole amounts of Cl- present in solutions at micromolar concentrations. This method was used to measure total intracellular chloride concentration ([Cl-]i) in leukocytes. Through the use of submicroliter samples, [Cl-] can be measured in the range of 5-500 microM. There is no measurable interference from other ions normally present in the cell and no intracellular ion is falsely measured as Cl-. [Cl-]i determined by the conventional coulometric titration and the new microelectrometric titration method was the same. Among the commonly used substituting anions, thiocyanate was the only one falsely measured as Cl- and should be avoided in experiments using this method. Because only picomole amounts of Cl- are required, measurements of intracellular pH and total concentrations of other intracellular ions can be done in the same lysate of as few as 5 X 10(5) cells. This feature should make it possible to study the time course of changes in [Cl-]i together with measurements of other intracellular ions following various physiological or experimental maneuvers.

Recombinant human interleukin 1-stimulated Na+/H+ exchange is not required for differentiation in pre-B lymphocyte cell line, 70Z/3.

Interleukin-1 a polypeptide hormone produced by activated macrophages is a mixture of at least two proteins, interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta). We have previously shown that macrophage-derived interleukin-1 induced new kappa light chain synthesis for surface IgM expression in a murine pre-B like cell line 70Z/3, a finding associated with an early amiloride-sensitive rise in the total intracellular sodium concentration. Because IL-1 alpha and IL-1 beta are structurally quite different, in this study their effect on 70Z/3 was examined separately. The results show that both human rIL-1 alpha and rIL-1 beta induce the differentiation of 70Z/3, but a higher concentration of rIL-1 beta compared to rIL-1 alpha is needed for a maximal response. At saturating concentrations, both rIL-1 alpha and rIL-1 beta induce a simultaneous rise in intracellular pH and sodium concentration. Because rIL-1 mediated intracellular alkalinization and sodium rise are amiloride sensitive, they likely occur through stimulation of the Na+/H+ exchanger across the cell membrane. Inhibition of the Na+/H+ antiport with an amiloride analog did not have an effect on rIL-1 induced surface IgM expression or the rIL-1-mediated increase in kappa light chain specific mRNA level. Therefore, these results indicate that an increase in pHi or [Na]i is not required for IL-1 induced 70Z/3 differentiation.

HCO3 accumulation in proximal tubule: roles of carbonic anhydrase, luminal buffers, and pH.

Most of filtered bicarbonate is reabsorbed in the early proximal tubule, and the high blood-to-lumen HCO3 concentration gradient generated is then maintained in the distal proximal tubule. To determine the factor(s) that prevent reaccumulation of HCO3 in the lumen, surface proximal tubules of the rat kidney were perfused in vivo. All perfusion solutions were similar in ionic composition to late proximal tubule fluid but, instead of HCO3, contained sulfate (SO4) or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Bicarbonate concentration ([HCO3]L) was measured by microcalorimetry, while collected fluid PCO2 was maintained in vitro at either renal cortical or atmospheric levels. A new single microelectrode was used to simultaneously measure PCO2 and luminal pH (pHL). With SO4 solution, pHL was 6.84 +/- 0.06, PCO2 was 50.8 +/- 5.0 mmHg, and [HCO3]L was 8.7 +/- 0.9 mM. When 10(-3) M acetazolamide (ATZ) was added to the perfusate, pHL was 7.00 +/- 0.08, PCO2 remained unchanged, [HCO3]L was 12.1 +/- 1.3 mM, and the rate of HCO3 accumulation increased by approximately 50%. When SO4 was replaced with HEPES, pHL increased to 7.18 +/- 0.05, PCO2 was unchanged, [HCO3]L was 16.5 +/- 1.4 mM, and the rate of HCO3 accumulation doubled. Because measured [HCO3]L and that calculated from pHL and PCO2 were approximately the same, the net gain of HCO3 occurred in vivo, a result of either HCO3 backflux and/or intraluminal generation from CO2. By allowing the collected fluid PCO2 to fall to near zero prior to [HCO3]L measurement, we estimate that approximately 50% of the total gain of HCO3 with all solutions was due to backflux.(ABSTRACT TRUNCATED AT 250 WORDS)

A new microelectrode method for simultaneous measurement of pH and PCO2.

A liquid-membrane pH-PCO2 microelectrode is described for the simultaneous measurement of pH and PCO2 in tissues and body fluids. The microelectrode is simple and easy to fabricate. It can be used to measure HCO3 concentration in solutions at chemical equilibrium. In the physiological range the microelectrode response is linear with nearly Nernstian slopes for PCO2 (61.2 +/- 2.0 mV/log10 PCO2) and pH (63.7 +/- 1.9 mV/pH units) (n = 14) at 37 degrees C. The PCO2 response is independent of the solutions' pH, anionic composition, and presence of serum proteins. In randomly micropunctured rat surface proximal tubules, pH averaged 6.80 +/- 0.04 and PCO2 averaged 57.7 +/- 4.6 mmHg (n = 22), whereas in the adjacent peritubular capillaries pH was higher (7.27 +/- 0.03) but PCO2 was not different (55.7 +/- 4.6 mmHg) (n = 22). Systemic arterial PCO2 was significantly lower compared with the renal cortex and averaged 37.4 +/- 2.4 mmHg (n = 14). Directly measured and pH-PCO2 microelectrode-derived HCO3 concentrations in systemic arterial blood, surface fluid bathing the kidney, and randomly micropunctured proximal tubules were approximately equal.