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S K Hanks

Publications and source records attributed to S K Hanks.

At least 37 records · Page 2Linked to original sources
PubMed1995

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.

3T3 Cells↗
PubMed1995

Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification.

The eukaryotic protein kinases make up a large superfamily of homologous proteins. They are related by virtue of their kinase domains (also known as catalytic domains), which consist of approximately 250-300 amino acid residues. The kinase domains that define this group of enzymes contain 12 conserved subdomains that fold into a common catalytic core structure, as revealed by the 3-dimensional structures of several protein-serine kinases. There are two main subdivisions within the superfamily: the protein-serine/threonine kinases and the protein-tyrosine kinases. A classification scheme can be founded on a kinase domain phylogeny, which reveals families of enzymes that have related substrate specificities and modes of regulation.

Amino Acid Sequence↗
PubMed1994

Focal adhesion kinase is abundant in developing blood vessels and elevation of its phosphotyrosine content in vascular smooth muscle cells is a rapid response to angiotensin II.

Focal adhesion kinase (FAK) is a structurally unique nonreceptor protein-tyrosine kinase that localizes to focal adhesion plaques. Regulation of its activity has been implicated in diverse signaling pathways, including those mediated by extracellular matrix/integrin interactions, G-protein coupled receptors for mitogenic neuropeptides, and certain oncogene products. To gain evidence for specific processes in which FAK may be involved in vivo, a study was initiated to determine its expression pattern during mouse development. FAK expression was detected in early embryos and appeared to be distributed throughout all cell types at about the time of neurulation. Subsequent to neural tube closure, expression became particularly abundant in the developing vasculature. This included expression in the medial layer of arteries populated by smooth muscle cells. In vitro studies using cultured rat aortic vascular smooth muscle cells demonstrate that FAK phosphotyrosine content is dramatically elevated in response to plating cells onto the adhesive glycoprotein, fibronectin. Also, enhanced tyrosine phosphorylation of FAK is observed in these cells upon stimulation with the vasoconstrictor angiotensin II. Thus, in vascular smooth muscle cells, like fibroblasts, FAK appears to play a role in signaling mechanisms induced by extracellular matrix components as well as G-protein coupled receptor agonists. The combined results of this study suggest that signaling through FAK may play an important role in blood vessel morphogenesis and function.

Angiotensin II↗
PubMed1994

Potential role for focal adhesion kinase in migrating and proliferating keratinocytes near epidermal wounds and in culture.

In normal, differentiating skin, hemidesmosomes make the stable attachment of basal epidermal keratinocytes to the dermis by linking the cytoplasmic keratin intermediate filaments to components of the basal lamina. In contrast, laterally migrating and proliferating basal keratinocytes in culture and presumably in repairing wounds use focal adhesions to form dynamic attachments to the dermis by linking actin microfilaments to the extracellular matrix. Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase concentrated along with phosphotyrosine-containing proteins in the focal adhesions of some cultured cells, is activated in vitro when cells attach, form focal adhesions, and spread. This report finds that FAK is activated, as determined from its increased phosphotyrosine content and from its increased labeling with [gamma-32P]ATP, in immunoprecipitates from human cultured keratinocytes attached and spreading on fibronectin compared to those attached but not spreading on polylysine. Furthermore, immunofluorescence shows that both FAK and phosphotyrosine are concentrated in the focal adhesions of cultured keratinocytes attached and spreading on extracellular matrix components known to facilitate cellular migration (fibronectin, collagens I or IV, and epiligrin). Finally, immunohistochemistry localizes FAK to the epidermal-dermal junction in repairing partial thickness burn wounds. FAK is found at the epidermal-dermal junction at sites and times which coincide with actively migrating or rapidly proliferating basal keratinocytes, suggesting that this distribution represents FAK concentrated and activated in adhesions analogous to the focal adhesions seen in cultured cells. Hence, FAK appears to have an important in vivo role in the reepithelialization of human wounds.

Burns↗
PubMed1993

Chicken and mouse focal adhesion kinases are similar in structure at their amino termini.

A novel protein-tyrosine kinase, designated 'Focal Adhesion Kinase' (FAK), has recently been implicated in signal transduction pathways activated by extracellular adhesion molecules and by neuropeptide growth factors. Previously deduced primary structures for chicken and mouse FAK polypeptides differ at their amino-termini, with mouse FAK reported to have a 25 amino acid residue extension not present in chicken FAK. Additional sequence information from the 5'-end region of the chicken FAK transcript now indicates that the amino-terminal extension previously thought to be unique to mouse FAK is, in fact, also predicted for chicken FAK. Thus mouse and chicken FAK polypeptides appear to be structurally similar throughout their lengths. This is further supported by comparison of their electrophoretic mobilities.

Amino Acid Sequence↗
PubMed1993

Focal-adhesion components are enriched in ventral membranes isolated from transformed keratinocytes in culture.

The lack of procedures for isolating or reconstituting intact focal adhesions has hindered studies of how focal adhesions are organized and of how their assembly/disassembly is controlled. A method for isolating large quantities of the ventral portion of plasma membranes from transformed keratinocytes (A-431 cells) in culture is described. Plasma membranes are stabilized using Zn2+ and the ventral portion isolated attached to the culture substratum after the body of the cell has been sheared away. Compared with complete plasma membranes isolated from cells scraped from the dish, these ventral-membrane preparations are enriched 18.5-fold and 5.1-fold in the focal-adhesion components talin and vinculin respectively. While the epidermal-growth-factor receptor-kinase is less abundant in preparations of ventral membranes, a recently described tyrosine kinase which localizes to focal adhesions in mouse fibroblasts is enriched 19.9-fold. Extracellular matrix components, as well as their integrin receptors, are also enriched in these preparations of the ventral portion of plasma membranes compared with preparations of complete plasma membranes.

Animals↗
PubMed1992

Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins.

Murine D type cyclins associate with a catalytic subunit (p34PSK-J3) with properties distinct from known cyclin-dependent kinases (cdks). Mouse p34PSK-J3 shows less than 50% amino acid identity to p34cdc2, p33cdk2, and p36cdk3, lacks a PSTAIRE motif, and does not bind to p13suc1. Cyclin D1-p34PSK-J3 complexes accumulate in macrophages during G1 and decline in S phase, whereas complexes involving cyclins D2 and D3 form in proliferating T cells. Although histone H1 kinase activity is not detected in cyclin D or PSK-J3 immunoprecipitates, cyclin D-p34PSK-J3 complexes assembled in vitro stably bind and phosphorylate the retinoblastoma gene product (pRb) and an Rb-like protein (p107) but do not interact with pRb mutants that are functionally inactive. Thus, p34PSK-J3 is a cyclin D-regulated catalytic subunit that acts as an Rb (but not H1) kinase.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1989

Molecular cloning of plant transcripts encoding protein kinase homologs.

Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences.

Amino Acid Sequence↗
PubMed1989

Messenger ribonucleic acid encoding an apparent isoform of phosphorylase kinase catalytic subunit is abundant in the adult testis.

The complete amino acid sequence for a novel member of the protein kinase family was deduced from the nucleotide sequence of a cloned human cDNA. This putative protein kinase, given the preliminary designation "PSK-C3," is similar in primary structure to phosphorylase kinase catalytic subunit (PhK-gamma) isolated from rabbit skeletal muscle. The level of similarity does not appear sufficient, however, to suggest that PSK-C3 represents the human homolog of skeletal muscle PhK-gamma. Rather, it seems likely that PSK-C3 is a novel PhK-gamma isoform. From a cross-species Northern hybridization experiment using adult rat tissue RNA, a transcript homologous to PSK-C3 was found to be abundant in the testis but could not be detected in any of 12 other tissues tested, including skeletal muscle, liver, and ovary. Increasing levels of PSK-C3 mRNA in the testis correlate with postnatal testicular development, suggesting possible hormonal regulation of gene transcription. Energy released by glycogeneolysis in the testis may help fuel the process of spermatogenesis.

Amino Acid Sequence↗
PubMed1988

The protein kinase family: conserved features and deduced phylogeny of the catalytic domains.

In recent years, members of the protein kinase family have been discovered at an accelerated pace. Most were first described, not through the traditional biochemical approach of protein purification and enzyme assay, but as putative protein kinase amino acid sequences deduced from the nucleotide sequences of molecularly cloned genes or complementary DNAs. Phylogenetic mapping of the conserved protein kinase catalytic domains can serve as a useful first step in the functional characterization of these newly identified family members.

Amino Acid Sequence↗
PubMed1988

Amino acid sequence of the BSC-1 cell growth inhibitor (polyergin) deduced from the nucleotide sequence of the cDNA.

The complete amino acid sequence of the BSC-1 cell growth inhibitor, including its precursor polypeptide, is reported. The sequence was deduced from the nucleotide sequence of the cDNA. The N-terminal amino acid sequence of the mature bioactive BSC-1 cell growth inhibitor is identical with the N-terminal sequences of the factors that have been called type beta 2 transforming growth factor and cartilage-inducing factor B, suggesting that these are identical. The complete amino acid sequence of the mature BSC-1 cell growth inhibitor differs from that of human type beta transforming growth factor in 32 of the 112 amino acids. Polyergin is proposed as the name for the BSC-1 cell growth inhibitor.

Amino Acid Sequence↗
PubMed1987

Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family.

Mixed oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identity with rabbit skeletal muscle phosphorylase kinase gamma-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.

Amino Acid Sequence↗
PubMed1986

Selective insolubility of active hsp70 gene chromatin.

A gentle chromatin fractionation procedure was used to investigate solubility properties of Drosophila hsp70 heat-shock genes. After a brief digestion of isolated nuclei with micrococcal nuclease, most DNA is readily solubilized under low-ionic-strength conditions that maintain native nucleosomal organization. Actively transcribing hsp70 genes, however, are found to be enriched in the insoluble nuclear residue. Inactive genes are not resistant to solubilization, showing a fractionation pattern similar to that of bulk DNA. The insolubility characteristic correlates well with two other structural features of active hsp70 chromatin: increased sensitivity to endonuclease attack and disruption of the nucleosomal repeat pattern. The 5'-flanking regulatory region of active hsp70 genes is particularly resistant to solubilization, suggesting a role for binding of transcription factors in mediating this effect.

Animals↗