To understand the regulation and function of Pyk2, we employed a yeast two-hybrid screen to identify novel proteins that interact with Pyk2. Since the full-length Pyk2 activated the Gal4 promoter for the reporter gene LacZ
when used as a bait (data not shown), we subcloned a cDNA fragment encoding the NH2
-terminal and kinase domains of Pyk2 (NKPyk2) into the pAS2 bait vector. Screening 4 × 106
clones of a HeLa cell library (Hannon et al. 1993
) yielded five clones that specifically interacted with NKPyk2. These interactions were confirmed by cotransforming yeast cells with the recovered prey plasmids and pAS2-NKPyk2, or by cotransforming the pAS2 vector alone as a control. Partial sequencing indicated that three clones encoded portions of a protein identical to a human gene KIAA0203 with an open reading frame of 1,591 residues. KIAA0203 is widely expressed in various human tissues, but its functions remain obscure ( A; Nagase et al. 1996
Based on its interaction with both Pyk2 and FAK as well as its apparent molecular mass (see below), we designated the full-length protein as FIP200 and the longest insert (encoded by pGAD33) of the three clones as CT-FIP (COOH-terminal FIP200). Analysis of the FIP200 protein sequence suggested it to be a cytoplasmic protein (e.g., no signal peptide for secretion, no transmembrane domain, and no nuclear localization signals). Residues 1,085–1,225 showed a high sequence homology (84% identity) with the mouse coiled-coil protein 1 and, therefore, was designated as a CC1-like region. In addition, a leucine zipper segment was found between residues 1,371 and 1,391 of FIP200 ( A).
To determine if FIP200 could bind directly to Pyk2 in vitro, a GST fusion protein containing CT-FIP (GST-CT-FIP) was prepared and tested for its binding to recombinant Pyk2 expressed in mammalian cells. B shows that Pyk2 bound GST-CT-FIP (middle lane), but not GST alone (left lane). To detect FIP200 association with Pyk2 in intact cells, 293T cells were cotransfected with pKH3-Pyk2 encoding HA-tagged Pyk2 and expression vectors encoding Flag-tagged FIP200, CT-FIP, and NH2-terminal FIP200 (NT-FIP; residues 1–641), or the vector alone as a control. Coimmunoprecipitation of the lysates showed that Pyk2 was associated with CT-FIP and FIP200, but not NT-FIP, in intact cells ( C). Western blotting of the immunoprecipitates with anti-Flag showed expression levels of FIP200 and its fragments ( D). Direct analysis of the lysates by Western blotting with anti-Pyk2 confirmed comparable expression of Pyk2 in all samples ( E). Consistent with the in vitro binding and transfection studies, association of endogenous Pyk2 and FIP200 was also detected by coimmunoprecipitation ( F). Together, these results demonstrated Pyk2 interaction with FIP200, and indicated that this interaction is mediated by the FIP200 COOH-terminal region.
Pyk2 and FAK are closely related tyrosine kinases that share similar structural organization and significant sequence homology (Avraham et al. 1995
; Lev et al. 1995
; Sasaki et al. 1995
). Therefore, we also examined the possibility of FIP200 association with FAK by both in vitro binding and in vivo coimmunoprecipitation experiments. A shows that FAK bound to GST-CT-FIP200 (middle lane), but not GST alone (left lane). To detect in vivo association, 293T cells were cotransfected with pKH3-FAK and pSG5-FIP200, pSG5-CT-FIP, or pSG5-Flag vector alone as a control. 2 d after transfection, cell lysates were prepared and immunoprecipitated with anti-Flag. B shows that FAK was associated with both the full-length (left lane) and COOH-terminal fragment of FIP200 (middle lane), but was absent in the immune complex from cells cotransfected with the control vector alone (right lane). Together, these results suggested that FIP200 may also associate with FAK via its COOH-terminal region.
Figure 2 FIP200 association with FAK. (A) Immobilized GST-CT-FIP or GST alone was incubated with lysates from 293T cells that had been transfected with pKH3-FAK. After washing, the bound proteins were analyzed by Western blotting with anti-HA. An aliquot of the (more ...)
We also examined subcellular localization of FIP200 first by using epitope tag antibodies (). Immunofluorescent staining of Flag-tagged FIP200 with anti-Flag showed a predominantly diffuse cytoplasmic distribution of FIP200 (A). Costaining with antivinculin verified the absence of FIP200 in focal contacts (B). Extensive colocalization of FIP200 with Pyk2 in the cytoplasm was confirmed by double label immunofluorescent staining of cells that had been cotransfected with Flag-tagged FIP200 and HA-tagged Pyk2 (C and D). We also attempted to detect localization of endogenous FIP200 using anti-FIP200, although our current anti-FIP200 did not work as well as the anti-epitope antibodies for immunofluorescent staining. Consistent with results using the anti-epitope antibodies, these studies suggested a cytoplasmic localization of endogenous FIP200 (data not shown). FIP200's predominantly cytoplasmic localization is consistent with its interaction with the cytoplasmic kinase Pyk2, although this does not exclude the possibility of its interaction with FAK (a fraction may be present in the cytoplasm, or even FAK in the focal contacts but facing the cytoplasm).
Figure 3 Subcellular localization of FIP200. NIH3T3 cells were transfected with vector encoding Flag-FIP200 (A and B) or both vectors encoding Flag-FIP200 and HA-Pyk2 (C and D). 1 d after transfection, cells were processed for immunofluorescent staining as described (more ...)
To examine the FIP200 binding site on Pyk2, we tested the potential interaction of CT-FIP with several fragments of Pyk2 in the yeast two-hybrid system (). As expected, NKPyk2 interacted with CT-FIP, which is encoded by pGAD33, but not the pGAD vector alone. The NH2-terminal domain of Pyk2 (NPyk2) did not exhibit interaction with CT-FIP or the control. In contrast, the fragment containing the kinase and COOH-terminal domains of Pyk2 (KCPyk2) exhibited a strong interaction with CT-FIP, although it also induced a slight basal activation when cotransformed with the control vector. Together, these data suggested that FIP200 binds to the kinase domain of Pyk2.
Specific Interaction of Pyk2 with CT-FIP in Yeast Two-Hybrid System
Association of FIP200 with Pyk2 at the kinase domain suggested that it may function as a regulator of the kinase. To test this possibility directly, Pyk2 was immunoprecipitated from RASM cells that had been stimulated by sorbitol for 5 min (Zheng et al. 1998
). They were subjected to in vitro kinase assays using E4Y1 as an exogenous substrate in the presence of different amounts of purified GST-CT-FIP or GST alone as controls. As shown in A, GST-CT-FIP inhibited Pyk2 kinase activity in a dose-dependent manner. However, comparable amounts of GST did not affect Pyk2 activity. Quantitative analysis of three independent experiments showed that Pyk2 kinase activity was inhibited up to ~80% at high doses of GST-CT-FIP. These results suggested that FIP200 might function as an inhibitor of Pyk2 by binding to its kinase domain directly.
Figure 4 Inhibition of Pyk2 kinase activity by CT-FIP. (A) Pyk2 was immunoprecipitated from RASM cell lysates. Aliquots of the immune complex were assayed for kinase activity using E4Y1 as an exogenous substrate in the presence of various amounts of GST-CT-FIP (more ...)
Pyk2 has been shown to bind Src family kinases (Dikic et al. 1996
). Therefore, it is possible that FIP200 reduced E4Y1 phosphorylation by affecting the association or activity of Src family kinases in the Pyk2 immune complex. To examine this possibility, we first tested the effects of the COOH-terminal FIP200 on the kinase activity of Pyk2 from SYF cells that are deficient in the expression of Src family kinases Src, Yes, and Fyn (Klinghoffer et al. 1999
). B shows that GST-CT-FIP inhibited the kinase activity of the Pyk2 isolated from these cells in a dose-dependent manner, whereas GST alone had little effect. Consistent with this, GST-CT-FIP inhibited the kinase activity of the Pyk2 mutant Y402F (lacking the binding site for Src family kinases) to a similar level as that for the wild-type Pyk2 ( C). Together, these results suggested that FIP200 inhibited Pyk2 kinase activity itself rather than the binding or activity of the associated Src family kinases. Similar experiments indicated that GST-CT-FIP could also inhibit FAK kinase activity in vitro ( D).
To determine whether binding of FIP200 to Pyk2 inhibited its kinase activity in intact cells, expression vectors encoding Pyk2 and FIP200 or its fragments were cotransfected into CHO cells. 2 d after transfection, the cells were treated with sorbitol and the lysates were prepared from these cells. HA-tagged Pyk2 was immunoprecipitated with anti-Pyk2 and analyzed for its tyrosine phosphorylation by Western blotting with antiphosphotyrosine mAb PY20. A shows that tyrosine phosphorylation of Pyk2 was significantly increased by sorbitol treatment, as observed previously (top; Zheng et al. 1998
). Cotransfection with CT-FIP reduced the increase in Pyk2 phosphorylation, whereas coexpression of the pSG5 vector had no effect. Western blotting of the immunoprecipitates with anti-Pyk2 verified similar levels of Pyk2 expression in these samples (middle). Western blotting with anti-Flag confirmed the expression of CT-FIP in the cotransfection samples (bottom). Similar studies indicated that cotransfection of the FIP200 ( B) but not the NT-FIP fragment ( C), also inhibited Pyk2 phosphorylation in response to sorbitol. Together, these results indicated that FIP200 could function as an inhibitor of Pyk2 in intact cells, and the inhibitory activity correlated with FIP200 binding to Pyk2 via its COOH-terminal fragment.
Figure 5 FIP200 inhibition of Pyk2 tyrosine phosphorylation in response to sorbitol. CHO cells were cotransfected with pKH3-Pyk2 and pSG5 vectors encoding FIP200, NT-FIP, CT-FIP, or pSG5 alone, as indicated. 2 d after transfection, cells were serum-starved for (more ...)
A variety of biological stimuli have been shown to activate Pyk2 in different cells (Lev et al. 1995
; Dikic et al. 1996
; Tokiwa et al. 1996
; Yu et al. 1996
). Thus, our results of FIP200 functioning as an inhibitor of Pyk2 also suggested the possibility that some of the activators of Pyk2 may activate the kinase by decreasing association of FIP200 with Pyk2. To test this possibility, we examined the association of the endogenous FIP200 and Pyk2 in cells that had been treated with various agents, which have been shown to stimulate Pyk2 (Zheng et al. 1998
). Consistent with previous results, shows that stimulation of RASM cells with PDGF, sorbitol, angiotensin II (AGII), or PMA all induced tyrosine phosphorylation of Pyk2 when compared with serum-starved or growing cells (A and B). Interestingly, a corresponding decrease in FIP200 association with Pyk2 was also observed upon stimulation with these agents (C and D). These results suggested that FIP200 dissociation from Pyk2 may mediate activation of Pyk2 by some biological stimuli. They also provided further support for our hypothesis that FIP200 functions as a Pyk2 inhibitor in intact cells.
Figure 6 Pyk2 phosphorylation and its association with FIP200 in response to different stimuli. Serum-starved RASM cells were stimulated with various agents as indicated (PDGF, 25 ng/ml for 5 min; sorbitol, 400 mM for 10 min; AGII, 1 μM (more ...)
It has been observed recently that transient transfection of Pyk2 induced apoptosis in a number of cell lines such as Rat1 cells, suggesting a possible role for Pyk2 in the regulation of apoptosis under certain conditions (Xiong and Parsons 1997
). To investigate the cellular function of FIP200 interactions with Pyk2, we examined the effects of FIP200 on induction of apoptosis by Pyk2 in Rat1 cells. Consistent with the previous results (Xiong and Parsons 1997
), transfection of Pyk2 into Rat1 cells resulted in the apoptosis of a large fraction of cells. Expression of FIP200 or its fragments alone had no statistically significant effects on apoptosis of Rat1 cells ( A). Interestingly, however, coexpression of FIP200 or CT-FIP with Pyk2 reduced induction of apoptosis by Pyk2 in these cells. In contrast, coexpression of NT-FIP did not affect the fraction of apoptotic cells induced by Pyk2 ( B). Therefore, FIP200 inhibition of Pyk2-induced apoptosis also correlated with its binding to Pyk2 () and its inhibition of Pyk2 kinase activity in intact cells (). Together, these results demonstrated FIP200 as an inhibitor of Pyk2 in the regulation of apoptosis by directly binding to the kinase.
Figure 7 FIP200 inhibition of Pyk2-induced apoptosis. (A) Rat-1 cells were cotransfected with a plasmid encoding GFP and expression vectors encoding Pyk2, FIP200, NT-FIP, CT-FIP, or vector alone (control), as indicated. The results show the mean and SD of a percentage (more ...)