Focal adhesion kinase (FAK) is a major mediator of integrin signaling pathways. The mechanisms of regulation of FAK activity and its associated cellular functions are not very well understood. Here, we present data suggesting that a novel protein FIP200 functions as an inhibitor for FAK. We show the association of endogenous FIP200 with FAK, which is decreased upon integrin-mediated cell adhesion concomitant with FAK activation. In vitro- and in vivo-binding studies indicate that FIP200 interacts with FAK through multiple domains directly. FIP200 bound to the kinase domain of FAK inhibited its kinase activity in vitro and its autophosphorylation in vivo. Overexpression of FIP200 or its segments inhibited cell spreading, cell migration, and cell cycle progression, which correlated with their inhibition of FAK activity in vivo. The inhibition of these cellular functions by FIP200 could be rescued by coexpression of FAK. Last, we show that disruption of the functional interaction between endogenous FIP200 with FAK leads to increased FAK phosphorylation and partial restoration of cell cycle progression in cells plated on poly-L-lysine, providing further support for FIP200 as a negative regulator of FAK. Together, these results identify FIP200 as a novel protein inhibitor for FAK.

Pyk2 is a recently described cytoplasmic tyrosine kinase that is related to focal adhesion kinase (FAK) and can be activated by a variety of stimuli that elevate intracellular calcium. In this report, we showed that Pyk2 and FAK tyrosine phosphorylation are regulated differentially by integrin-mediated cell adhesion and soluble factors both in rat aortic smooth muscle cells, which express endogenous Pyk2 and FAK, and in transfected Chinese hamster ovary cells. We also found that Pyk2 is diffusely present throughout the cytoplasm, while FAK is localized in focal contacts as expected, suggesting that the different localization may account for their differential regulation. By analyzing a chimeric protein contain N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, we provided evidence that the distinctive C-terminal domains of Pyk2 and FAK were responsible for their differential regulation by integrins and soluble stimuli as well as their subcellular localization. Finally, we correlated FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, with their regulation by integrins and focal contact localization. These results demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer their differential regulation by different subcellular localization and association with the cytoskeletal protein talin.Proline-rich tyrosine kinase 2 (Pyk2 1 ; also known as CAK␤, RAFTK, and CADTK) is a recently described cytoplasmic tyrosine kinase that is related to the focal adhesion kinase (FAK) (1-4). Recent studies have shown that Pyk2 is involved in calcium-induced regulation of ion channel and mitogen-activated protein kinase activation (1), stress-induced c-Jun Nterminal kinase activation (5), and Src-mediated activation of the mitogen-activated protein kinase signaling pathway in PC12 cells (6). Although these studies indicate that Pyk2 is involved in several signal transduction pathways, many questions concerning the regulation and function of Pyk2, especially the role of Pyk2 in cell adhesion, remain unanswered.Pyk2 and FAK share a similar structural organization with a tyrosine kinase domain flanked by non-catalytic domains at both the N and C termini. These two kinases are approximately 60% identical in the central catalytic domain and share approximately 40% identity in both the N-and C-terminal domains (1-3). Because of the high sequence homology and similar overall organization between Pyk2 and FAK, it is especially interesting to compare the regulation of Pyk2 with that of FAK, in particular their responses to integrin-mediated cell adhesion. Several recent reports have presented somewhat conflicting data regarding regulation of Pyk2 by integrin-mediated cell adhesion in different cell types. It has been reported that Pyk2 displays an integrin-dependent phosphorylation and is localized in focal contacts in B lymphocytes, megakaryocytes, and transfected COS cells (7,8); however, in transfected 3Y1 cells, Pyk2 phosphorylation is not increased upon plating on fibronectin (FN) an...

Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic tyrosine kinase implicated to play a role in several intracellular signaling pathways. We report the identification of a novel Pyk2-interacting protein designated FIP200 (FAK family kinase–interacting protein of 200 kD) by using a yeast two-hybrid screen. In vitro binding assays and coimmunoprecipitation confirmed association of FIP200 with Pyk2, and similar assays also showed FIP200 binding to FAK. However, immunofluorescent staining indicated that FIP200 was predominantly localized in the cytoplasm. FIP200 bound to the kinase domain of Pyk2 and inhibited its kinase activity in in vitro kinase assays. FIP200 also inhibited the kinase activity of the Pyk2 isolated from SYF cells (deficient in Src, Yes, and Fyn expression) and the Pyk2 mutant lacking binding site for Src, suggesting that it regulated Pyk2 kinase directly rather than affecting the associated Src family kinases. Consistent with its inhibitory effect in vitro, FIP200 inhibited activation of Pyk2 and Pyk2-induced apoptosis in intact cells, which correlated with its binding to Pyk2. Finally, activation of Pyk2 by several biological stimuli correlated with the dissociation of endogenous FIP200–Pyk2 complex, which provided further support for inhibition of Pyk2 by FIP200 in intact cells. Together, these results suggest that FIP200 functions as an inhibitor of Pyk2 via binding to its kinase domain.

NusA and Rho are essential Escherichia coil proteins that influence tanscription elongation and termination. We show that an E. coli derivative unable to express NusA, because its sole nusA gene contains a lrge deletion/substitution, is viable providing that the bacterium also carries a rho mutation that reduces tra iption termination.This Rho-miated suppression is not allee specific, since either a mutation changing amino acid 134 [rho(E134D)] or a mutation canging amino add 352 (rho)) allows growth of a nusA-deleted E. coil. However, both rho mutations similarly decrease transcription termination 8-to 9-fold. We propose that the essential role of NusA is to enhance pausing of RNA polymerase at certain sites, permitting tight coupling of transcription and translation. This coupling interferes with Rho access to and/or movement on the nascent RNA and blocks premature termination of transcription. Thus, NusAdependent coupling should be less important in a mutant with low Rho activity. The fact that E. coi grows without NusA argues that NusA should be considered an accessory factor rather than a subunit of RNA polymerase.

Sphingosine 1-phosphate (SphP), a metabolite of cellular sphingolipids, has been shown to induce cell proliferation by activating the mitogen-activated protein kinase (MAPK) pathway. Proline-rich tyrosine kinase 2 (Pyk2) is a novel cytosolic tyrosine kinase which mediates activation of the MAPK or c-Jun N-terminal kinase (JNK) signaling pathways in response to a variety of stimuli that elevate intracellular calcium. In this report, we show that SphP stimulates both tyrosine phosphorylation of Pyk2 and MAPK activation in a transient and dose-dependent manner in rat aortic smooth muscle cells. Further studies indicate that Pyk2 phosphorylation, but not MAPK activation, is dependent on a pertussis toxinsensitive G-protein-coupled receptor as well as partially on actin cytoskeleton. In addition, both intracellular calcium mobilization and protein kinase C (PKC) are required for optimal Pyk2 phosphorylation while either calcium increase or PKC activation is sufficient for MAPK activation in response to SphP. Finally, we show that a tyrosine kinase(s) other than Pyk2 is necessary for MAPK activation by SphP. Together, these results suggest that SphP stimulates tyrosine phosphorylation of Pyk2 through a G-protein coupled receptor, which is dissociated from its activation of the MAPK pathway in these cells.

The N gene product of coliphage gamma, with a number of host proteins (Nus factors), regulates phage gene expression by modifying RNA polymerase to a form that overrides transcription-termination signals. Mutations in host nus genes diminish this N-mediated antitermination. Here, we report the isolation and characterization of the rpoAD305E mutation, a single amino acid change in the carboxy terminal domain (CTD) of the alpha subunit of RNA polymerase, that enhances N-mediated antitermination. A deletion of the 3' terminus of rpoA, resulting in the expression of an alpha subunit missing the CTD, also enhances N-mediated antitermination and, similar to rpoAD305E, suppresses the effect of nus mutations. Thus, the N-Nus complex may be affected through contacts with the CTD of the alpha subunit of RNA polymerase, as is a group of regulatory proteins that influences initiation of transcription. What distinguishes our findings on the N-Nus complex from those of previous studies with transcription proteins is that all of the regulators characterized in those studies bind DNA and influence transcription initiation; whereas the N-Nus complex binds RNA and affects transcription elongation. A screen of some previously identified rpoA mutations that influence transcription activators revealed only one other amino acid change, L290H, in the CTD of the alpha subunit, that influences antitermination. Although our results provide evidence that interactions of the alpha subunit of RNA polymerase must be considered in forming models of transcription antitermination, they do not provide information as to whether the interactions of alpha that ultimately influence antitermination occur during initiation or during elongation of transcription.

The Escherichia coli nusA gene, nusA,E, encodes an essential protein that influences transcription elongation.Derivatives of E. coli in which the SalmoneUla typhimurium nusA gene, nusAs, has replaced nusA$4 are viable. The nusA gene of Escherichia coli encodes an essential 54,400-Mr protein that functions in transcription elongation (reviewed in references 20, 29, and 83) and termination (16,37,49,75,81). Consistent with these roles, NusA has been shown to influence pausing of RNA polymerase (16,17,47,50,75,76,85). The demonstration of an association between NusA and RNA polymerase (31,35,43,46,74) suggests that NusA may influence transcription by a direct interaction with polymerase.NusA was identified through its role in the regulated expression of phage X genes (reviewed in references 13, 20, 23, and 67). Early A transcription initiating at promoters PL and PR partially terminates at terminators tLl (15, 70) and tRl (10), respectively. The escaping transcripts in the latter case terminate completely at a collection of terminators in the nin region (8,9,11,48, 52) (Fig.

We have previously identified FAK and its associated signaling pathways as a mediator of cell cycle progression by integrins. In this report, we have analyzed the potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation by using tetracycline-regulated expression system as well as chimeric molecules. We have found that induction of Pyk2 inhibited G(1) to S phase transition whereas comparable induction of FAK expression accelerated it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increased cell cycle progression as FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibited cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicated that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 had little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibited Erk activation in cell adhesion whereas FAK and PFhy1 stimulated it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. Finally, we showed that while FAK and PFhy1 were present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) were tyrosine phosphorylated and associated with Src and Fyn. This suggested that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant (Δ)C14 described previously by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition.