Correlations between the expression, phosphotyrosine content and enzymatic activity of focal adhesion kinase, pp125FAK, in tumor and nontransformed cells.

Focal adhesion kinase (pp125FAK, FAK) is a structurally unique nonreceptor tyrosine kinase that is localized in the focal adhesion plaques. Activation or modulation of this kinase has been associated with several signaling pathways including integrin mediated processes, mitogenic stimulation by neuropeptides and platelet-derived growth factor as well as oncogene-mediated transformation. These observations suggest that FAK may play a potential role in tumorigenesis and/or tumor invasiveness. Since the phosphotyrosine content of FAK has been implicated in both the activation of its catalytic activity and the recruitment of SH2 containing proteins, the expression, phosphorylation status and enzymatic activity of FAK was examined in a number of human tumor and normal cell lines. FAK was detectable in all cell lines with fairly consistent levels of expression. In contrast, constitutive tyrosine phosphorylation of FAK was quite variable among both normal and tumor cell lines. A direct correlation (correlation coefficient = 0.94) was observed between FAK activity and phosphotyrosine content. Within the cell lines examined, colon carcinomas exhibited marked elevation in FAK tyrosine kinase activity and phosphotyrosine content. These data suggest that colon carcinomas have elevated FAK activity in comparison to other tumor types and provide further support that the catalytic activity of FAK is enhanced by its phosphotyrosine content.

Expression, purification and characterization of focal adhesion kinase using a baculovirus system.

Focal adhesion kinase (FAK) has been overexpressed in insect cells using a baculovirus expression system. A recombinant baculovirus was generated which contains the mouse FAK cDNA cloned into a histidine tag transfer vector. Synthesis of the immunoreactive recombinant protein (baculovirus focal adhesion kinase (BFAK) Mr approximately 125,000) in infected Sf9 cells was detected 23 h postinfection, with maximal accumulation occurring at 48 h postinfection. BFAK constituted 5.4% of total soluble protein in the insect cell lysate and represented 19 mg/liter culture (approximately 2 x 10(9) cells). The enzyme was active as a protein tyrosine kinase in both SF9 cells and in vitro. Purification to near homogeneity was achieved by nickel chelation chromatography. A yield of 5 mg of purified active BFAK was consistently produced from 1 liter of infected insect cells. BFAK tyrosine kinase activity was characterized physically using poly(Glu-Tyr) as a substrate. BFAK activity required the presence of a divalent cation and exhibited a preference for Mn2+ over Mg2+. Maximal tyrosine kinase activity was attained at pH 7.2. Steady-state kinetic analysis with respect to ATP concentration did not conform to simple Michaelis-Menten kinetics and exhibited a Hill coefficient of much less than 1. Km values for ATP using native and autophosphorylated BFAK were 6.7 +/- 1.0 and 4.3 +/- 0.2 microM, respectively. Kcat values were 13.9 +/- 1.9 and 8.9 +/- 0.3 nmol/min/mg BFAK. Steady-state kinetics with respect to the peptide substrate did fit the Michaelis-Menten equation and exhibited a Km value of 2.4 +/- 0.3 micro/ml.

DNA determinants in sequence-specific recognition by XmaI endonuclease.

The XmaI endonuclease recognizes and cleaves the sequence C decreases CCGGG. Magnesium is required for catalysis, however, the enzyme forms stable, specific complexes with DNA in the absence of magnesium. An association constant of 1.2 x 10(9)/M was estimated for the affinity of the enzyme for a specific 195 bp fragment. Competition assays revealed that the site-specific association constant represented an approximately 10(4)-fold increase in affinity over that for non-cognate sites. Missing nucleoside analyses suggested an interaction of the enzyme with each of the cytosines and guanines within the recognition site. Recognition of each of the guanines was also indicated by dimethylsulfate interference footprinting assays. The phosphates 5' to the guanines within the recognition site appeared to be the major sites of interaction of XmaI with the sugar-phosphate backbone. No significant interaction of the protein was observed with phosphates flanking the recognition sequence. Comparison of the footprinting patterns of XmaI with those of the neoschizomer SmaI (CCC decreases GGG) revealed that the two enzymes utilize the same DNA determinants in their specific interaction with the CCCGGG recognition site.

Sequence-specific DNA recognition by the SmaI endonuclease.

SmaI endonuclease recognizes and cleaves the sequence CCC decreases GGG. The enzyme requires magnesium for catalysis; however, equilibrium binding assays revealed that the enzyme binds specifically to DNA in the absence of magnesium. A specific association constant of 0.9 x 10(8) M-1 was determined for SmaI binding to a 22-base duplex oligonucleotide. Furthermore, the KA was a function of the length of the DNA substrate and the enzyme exhibited an affinity of 1.2 x 10(9) M-1 for a 195-base pair fragment and which represented a 10(4)-fold increase in affinity over binding to nonspecific sequences. A Km of 17.5 nM was estimated from kinetic assays based on cleavage of the 22-base oligonucleotide and is not significantly different from the KD estimated from the thermodynamic analyses. Footprinting (dimethyl sulfate and missing nucleoside) analyses revealed that SmaI interacts with each of the base pairs within the recognition sequence. Ethylation interference assays suggested that the protein contacts three adjacent phosphates on each strand of the recognition sequence. Significantly, a predicted protein contact with the phosphate 3' of the scissile bond may have implications in the mechanism of catalysis by SmaI.

The endonuclease isoschizomers, SmaI and XmaI, bend DNA in opposite orientations.

The SmaI and XmaI endonucleases are imperfect isoschizomers that recognize the sequence CCCGGG. SmaI cleaves between the internal CpG to produce blunt end scissions whereas XmaI cleaves between the external cytosines to produce a four base, five prime overhang. Each of the endonucleases forms stable, specific complexes with DNA in the absence of magnesium. Circular permutation analyses of the protein-DNA complexes revealed that each of the endonucleases induces bending of the DNA. Phase sensitive detection analyses verified the existence of the SmaI and XmaI induced bends. Furthermore, bending of the helix axis by the endonucleases appeared to be directed in opposite orientations. The orientation of the SmaI induced bend appeared to be towards the major groove and is reminiscent of the direction of the bend induced by EcoRV which similarly induces blunt end scissions. Conversely, XmaI appeared to bend the DNA towards the minor groove.

Structure and evolution of the XcyI restriction-modification system.

The XcyI restriction-modification system from Xanthomonas cyanopsidis recognizes the sequence, CCCGGG. The XcyI endonuclease and methylase genes have been cloned and sequenced and were found to be aligned in a head to tail orientation with the methylase preceding and overlapping the endonuclease by one base pair. The nucleotide sequence codes for an N4 cytosine methyltransferase with a predicted molecular weight of 33,500 and an endonuclease comprised of 333 codons and a molecular weight of 36,600. Sequence comparisons revealed significant similarity between the XcyI, CfrI and SmaI methylisomers. In contrast, no similarity was detected between the primary structures of the XcyI and SmaI endonucleases. The XcyI restriction-modification system is highly homologous to the XmaI genes, although the DNA sequences flanking the genes rapidly diverge. The sequence of the XcyI endonuclease contains two motifs which have recently been identified as essential to the activity of the EcoRV endonuclease.