A second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily, cell adhesion kinase ␤ (CAK␤), was identified by cDNA cloning. The rat CAK␤ is a 115.7-kDa PTK that contains N-and C-terminal domains of 418 and 330 amino acid residues besides the central kinase domain. The rat CAK␤ has a homology with mouse FAK over their entire lengths except for the extreme N-terminal 88 residues and shares 45% overall sequence identity (60% identical in the catalytic domain), which indicates that CAK␤ is a protein structurally related to but different from FAK. The CAK␤ gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAK␤ antibody immunoprecipitated a 113-kDa protein from rat brain, 3Y1 fibroblasts, and COS-7 cells transfected with CAK␤ cDNA. The tyrosinephosphorylated state of CAK␤ was not reduced on trypsinization, nor enhanced in response to plating 3Y1 cells onto fibronectin. CAK␤ localized to sites of cell-tocell contact in COS-7 transfected with CAK␤ cDNA, in which FAK was found at the bottom of the cells. Thus, CAK␤ is a PTK possibly participating in the signal transduction regulated by cell-to-cell contacts. Protein-tyrosine kinases (PTKs)1 that do not span the plasma membranes (so-called nonreceptor PTKs) have been classified into different subclasses (subfamilies) based on the sequence similarity and distinct structural characteristics (1). Many nonreceptor PTKs participate in cellular signal transduction by associating with the intracellular portions of transmembrane receptors which do not themselves have PTK activity. Different nonreceptor PTKs play diverse and specific roles in mediating the signal transduction by different nonkinase receptors (2-4).Focal adhesion kinase (FAK) has been proposed as the prototype (and hitherto the sole member) of a new subfamily of nonreceptor PTK, represented by proteins with large N-and C-terminal domains flanking the catalytic domain but without Src homology 2 and 3 (SH-2 and SH-3) domains (5-9). FAK is concentrated in focal adhesions (5, 6), and its phosphorylation and activation are triggered by the ligand binding to integrins and by the stimulation of certain growth factor and neuropeptide receptors (6, 10 -24). The N-and C-terminal domains of FAK mediate its interactions with integrins, the Src-family kinases and paxillin, a focal adhesion associated protein (8,9,(25)(26)(27)(28). By these and other yet to be characterized interactions, FAK regulates signaling via different receptors. Because only one member of the FAK subfamily is known to date, we sought to identify a second PTK of the FAK subfamily by a homologybased cDNA cloning strategy. We describe here an isolation and characterization of a cDNA coding for a new member of the FAK family. The novel PTK described here is the second member, to our knowledge, of the FAK subfamily whose cDNA has been cloned and sequenced and is designated CAK␤ for cell adhesion kinase ␤.

Long-term potentiation (LTP) is an activity-dependent enhancement of synaptic efficacy, considered a model of learning and memory. The biochemical cascade producing LTP requires activation of Src, which upregulates the function of NMDA receptors (NMDARs), but how Src becomes activated is unknown. Here, we show that the focal adhesion kinase CAKbeta/Pyk2 upregulated NMDAR function by activating Src in CA1 hippocampal neurons. Induction of LTP was prevented by blocking CAKbeta/Pyk2, and administering CAKbeta/Pyk2 intracellularly mimicked and occluded LTP. Tyrosine phosphorylation of CAKbeta/Pyk2 and its association with Src was increased by stimulation that produced LTP. Finally, CAKbeta/Pyk2-stimulated enhancement of synaptic AMPA responses was prevented by blocking NMDARS, chelating intracellular Ca(2+), or blocking Src. Thus, activating CAKbeta/Pyk2 is required for inducing LTP and may depend upon downstream activation of Src to upregulate NMDA receptors.

No abstract

The mechanisms by which stimuli that raise cytosolic free Ca 2؉ concentrations in neurons can increase protein tyrosine phosphorylation are not known. Using rat hippocampal slices and cortical synaptosomes, we have examined the regulation of two highly related cytoplasmic tyrosine kinases, pp125 focal adhesion kinase (pp125 FAK ) and proline-rich tyrosine kinase 2/cell adhesion kinase ␤ (PYK2/CAK␤ /calmodulin kinases (3), and of a serine/threonine phosphatase, calcineurin (4). In addition, cytosolic Ca 2ϩ is able to activate protein tyrosine phosphorylation and signaling cascades involving Ras and MAP kinase which result in alterations in gene transcription (see Ref. 5 for a review). Depolarization and neurotransmitter agonists increase tyrosine phosphorylation of several proteins in synaptosomes (6), hippocampal slices (7), neurons in culture (7), and cell lines with neuronal characteristics (8,9). This link between tyrosine phosphorylation and Ca 2ϩ in neurons opens exciting perspectives since tyrosine phosphorylation appears necessary for the establishment of LTP (10) and LTD (11) and plays an important role in the control of neuronal differentiation and survival (5). However, the molecular basis for the stimulation of tyrosine phosphorylation in response to Ca 2ϩ is not known. Evidences obtained in PC12 cells indicate that Src (8) and PYK2 (9) are involved in the Ca 2ϩ -induced activation of tyrosine phosphorylation and downstream signaling cascades. Yet, the mechanism by which Ca 2ϩ activates Src and PYK2 in PC12 cells is not known, and it is not clear which tyrosine kinase is activated first. In addition, the regulation of these, or related, kinases has not been demonstrated in normal neuronal tissue. Here, we have investigated, in rat hippocampus, the possible regulation by Ca 2ϩ of two structurally related cytoplasmic tyrosine kinases, pp125 FAK and PYK2/CAK␤, which could be associated with Src family kinases in neurons. pp125 FAK is a 125-kDa cytosolic tyrosine kinase devoid of SH2 or SH3 domains, which is associated with focal adhesions (12, 13). pp125FAK is phosphorylated on tyrosine in response to integrin engagement and to stimulation of various G proteincoupled receptors (14). Autophosphorylated pp125 FAK binds to the SH2 domain of Src or Fyn (15). Phosphorylation of pp125 FAK by Src on multiple residues (16) allows the recruitment and activation of phosphatidylinositol-3-kinase (17, 18) and the binding of Grb2, leading to the activation of MAP kinase cascade (19). pp125FAK is highly expressed in nervous tissue during development, a period at which it is enriched in neuronal growth cones (20). In the brain of adult rats, pp125FAK is expressed at higher levels than in most other tissues, especially in the hippocampus, and the cerebral cortex (20). However, pp125FAK immunoreactivity is rather diffuse in adult neurons (20), and its precise localization and function are not known. Interestingly, pp125FAK appears to be a major phosphoprotein altered in Fyn knock-out mice (21), which dis-* The costs of p...

pp125FAK and CAK␤/Pyk2/CadTK/RAFTK are related protein-tyrosine kinases. It is therefore of interest whether CAK␤ shares some of the properties of pp125 FAK . Using recombinant glutathione S-transferase fusion proteins, we show that the C-terminal domains of both proteins bind paxillin in vitro. The C-terminal domain of CAK␤ was engineered to be autonomously expressed in chicken embryo cells and, like pp125 FAK and p41/43 FRNK (the C-terminal noncatalytic domain of pp125 FAK ), was found to localize to cellular focal adhesions. In contrast, full-length CAK␤ was generally found diffusely distributed throughout the cell, although a fraction of the cells exhibited focal adhesion localization. Vanadate treatment of pp125 FAK -and CAK␤-overexpressing CE cells induced a dramatic increase in the phosphotyrosine content of a common set of proteins including tensin, paxillin, and p130Cas , but some of these substrates, particularly p130Cas , appeared to be differentially phosphorylated by pp125 FAK and CAK␤. Levels of tyrosine phosphorylation were higher in CAK␤-overexpressing cells, and additional phosphotyrosine-containing species were specifically immunoprecipitated. In addition, vanadate treatment of CE cells overexpressing CAK␤, but not pp125 FAK overexpressors, induced a profound morphological change, which could be a consequence of the observed differences in substrate phosphorylation. pp125 FAK , 1 the focal adhesion kinase, is a 125-kDa proteintyrosine kinase (PTK) that is discretely localized to cellular focal adhesions (1, 2). Thus, pp125FAK colocalizes with the integrins, which are heterodimeric, transmembrane receptors that bind to proteins in the extracellular matrix, e.g. fibronectin and collagen (3, 4). The integrins function in cell adhesion cytoskeleton anchorage and in the transduction of extracellular stimuli into cytoplasmic signals, including the phosphorylation of proteins on tyrosine (5, 6). pp125 FAK is one of the major substrates for integrin-dependent tyrosine phosphorylation, and concomitant with tyrosine phosphorylation, it becomes enzymatically active (2, 7-10). It is therefore anticipated that pp125 FAK will prove a fundamental element in an integrinregulated signaling pathway. In addition, a number of other stimuli, including neuropeptides and growth factors, have been reported to induce tyrosine phosphorylation of pp125 FAK (5). The function of pp125 FAK has been explored by disrupting endogenous pp125FAK signaling. Overexpression of the C-terminal noncatalytic domain of pp125 FAK , which contains the focal adhesion targeting sequence, blocks pp125 FAK -induced tyrosine phosphorylation of substrates and impairs the spreading of chicken embryo cells on fibronectin (11). Microinjection of a similar fragment of pp125 FAK into human umbilical vein endothelial cells had little effect upon the structure of focal adhesions but abolished focal adhesion-associated phosphotyrosine and inhibited cell migration (12). pp125 FAK expression has also been obliterated by gene knockout, which results in em...