PAK4 directly interacts with the integrin β5 subunit cytoplasmic domain
By means of yeast two-hybrid screening of a 19-d mouse embryo cDNA library and following remating tests, we identified six known or hypothetic proteins specifically interacting with the human integrin β5 cytoplasmic domain (). 25 clones were found to interact with the integrin β5 cytoplasmic domain. Sequence analysis revealed that six of these mouse cDNA clones encoded a sequence highly homologous to hPAK4 kinase domain (KD), and were therefore identified as mouse PAK4 (mPAK4), which strongly and specifically interacted with integrin β5 cytoplasmic domain in the repeated yeast mating tests (unpublished data). An aa sequence comparison of the KD of mPAK4 with hPAK4, hPAK1, and the Drosophila
PAK homologue mushroom body tiny gene product (MBT) (Melzig et al., 1998
) is shown in A. mPAK4 KD shares 98% homology in aa sequence with hPAK4 KD, 83% with MBT KD, and 56% with hPAK1. The interaction of PAK4 with integrin β5 was then further analyzed by independent biochemical methods both in vitro and in living cells.
In GST pull-down assays, we found an association of integrin β5 from cell lysates to a purified GST-fused PAK4 KD and of PAK4 to a purified GST-fused β5 cytoplasmic domain ( B). In addition, HA-tagged PAK4 was coimmunoprecipitated with integrins β3 and αvβ5 in living cells ( C, top). In C, the β3 immunoprecipitation (IP) appears to bring down more PAK4 than the IP for αvβ5. However, the expression levels of the two integrins are different (unpublished data) and the two antibodies used may be differently efficient for IP. Therefore, differences in PAK4 amounts in this IP cannot be used to indicate relative binding strengths. Furthermore, the reverse IP of HA–PAK4 brought down both integrin αv and β5 subunits from a cell lysate ( C, middle and bottom), whereas IP of an irrelevant HA-tagged p21CIP1
did not (unpublished data). Importantly, by IP we also found an association of endogenous PAK4 with endogenous integrin αvβ5 in living cells ( D).
Identified interactors with human integrin β5 subunit cytoplasmic domain by yeast two-hybrid screening
Figure 1. PAK4 interacts with the integrin β5 subunit. (A) Amino acid sequence comparison of mPAK4 KD, found by the yeast two-hybrid screening with hPAK4, Drosophila MBT (MBT), and hPAK1 KDs. Dashes stand for identical amino acids and the variations of (more ...)
PAK4 interacts with the membrane-proximal region of the integrin β5 cytoplasmic domain
To determine which region within the integrin cytoplasmic domain interacts with PAK4, we generated cDNAs encoding various regions of the cytoplasmic domain of integrin β5 by PCR and cloned them into the bait vector pEG202. Yeast mating experiments were performed using a prey vector that contains PAK4 KD (aa 239–591) and the various bait vectors. The PAK4-binding region was mapped to aa 759–767 within the integrin β5 cytoplasmic domain ( A). Furthermore, association of endogenous PAK4 to the membrane-proximal region of integrin β5 subunit was verified by a GST pull-down assay ( B), in which PAK4 associated with GST–β5 cytoplasmic domain, but not with a GST–β5 deletion mutant lacking the PAK4-binding region identified by yeast mating tests. Amino acid sequences of other integrin β subunits corresponding to the PAK4-binding region of integrin β5 cytoplasmic domain were aligned ( C), displaying a moderate sequence homology.
Figure 2. Mapping of the PAK4 binding region within the integrin β5 cytoplasmic domain. (A) Mapping of the PAK4-binding region in the integrin β5 cytoplasmic domain. Various regions of the integrin β5 cytoplasmic domain were cloned into (more ...)
Integrin β5 interacts with a PAK4 COOH-terminal region
To determine the region within PAK4 that interacts with the integrin β5 cytoplasmic domain, we generated cDNAs encoding various regions of the PAK4 KD that were amplified by PCR and cloned into the prey vector pJG4-5. Yeast mating experiments were performed using a bait vector that contains the integrin β5 cytoplasmic domain (aa 753–799) and the various prey vectors. We mapped the integrin-binding region to aa 505–530 within the PAK4 KD by yeast two-hybrid mating tests ( A), and further confirmed the requirement of this region of PAK4 for association with integrin αvβ5 in mammalian cells by IP using a PAK4 deletion mutant ( B). Therefore, we denote this region as the integrin-binding domain (IBD) in PAK4. The aa sequences of other PAK family members, including the Drosophila PAK4 homologue MBT, were aligned in comparison with the PAK4 IBD ( C). The IBD region is highly homologous among PAK family members, suggesting that family members other than PAK4 might also hold the capacity to bind to integrin cytoplasmic domains. In addition, a schematic illustration of known PAK4 functional motifs indicates the location of IBD within the PAK4 KD ( D).
Figure 3. Mapping of the integrin β5–binding region within the PAK4 KD. (A) Various regions of the PAK4 KD were cloned into the prey vector pJG4-5 and then mated in a yeast two-hybrid assay with the integrin β5 cytoplasmic domain in the (more ...)
Translocation of PAK4 to lamellipodia by integrin ligation to VN and colocalization with integrin αvβ5
Given that PAK4 associates with integrin αvβ5, we analyzed the effect on cellular distribution of endogenous PAK4 by αvβ5-mediated attachment to VN in MCF-7 cells, which exclusively use integrin αvβ5 for attachment to VN (unpublished data). Before replating, we observed a cytosolic distribution of PAK4 in MCF-7 cells under normal culture conditions ( A). We then examined the endogenous PAK4 distribution after replating cells onto VN. Interestingly, we found a remarkable redistribution of PAK4 to forming lamellipodial structures in the cellular periphery as early as 10 min after replating on VN. With longer cell attachment, PAK4 was distributed into membrane ruffles and leading edges. However, cells replated onto poly-l-lysine (PLL) that are attached in an integrin-independent manner remained unspread with PAK4 distributed in the cytosol ( A). The fact that attachment to VN is mediated by integrin αvβ5, whereas attachment to PLL is integrin independent, indicates that integrin ligation may specifically stimulate relocalization of PAK4 to lamellipodia. Importantly, the redistribution of PAK4 upon cell attachment may allow PAK4 to associate with integrins in lamellipodia, which are sites of integrin attachment to the underlaying ECM. In addition, we observed a similar relocalization of PAK4 in M21 human melanoma cells and ECV 304 human bladder carcinoma cells (unpublished data), suggesting that the relocalization of PAK4 upon replating onto VN occurs in various cell types.
Figure 4. Relocalization of PAK4 to the cell membrane and colocalization of PAK4 and integrin αvβ5 in MCF-7 cells. (A) MCF-7 human breast carcinoma cells, under normal culture conditions or replated onto VN or PLL for indicated times, were stained (more ...)
To examine whether the lamellipodia-localized PAK4 could physically meet with integrin αvβ5 at the cell membrane, we replated MCF-7 cells onto VN and costained the cells for endogenous PAK4 and endogenous integrin αvβ5. As shown in B, PAK4 and integrin αvβ5 colocalized in lamellipodia shortly after replating onto VN. PAK4 may therefore be able to engage in integrin-mediated cellular functions.
Relocalization of PAK4 to lamellipodia does not require its kinase activity, integrin interaction, or Cdc42/Rac binding
Given that PAK4 undergoes membrane relocalization upon attachment onto VN, it was of interest to elucidate whether the relocalization of PAK4 is dependent on its integrin or Cdc42/Rac interaction and/or its kinase activity. Therefore, we constructed Flag-tagged PAK4 mutants that lack the binding capacity for Cdc42/Rac (PAK4-L19, 22), the IBD (PAK4-ΔIBD), or PAK4 kinase activity (PAK4-M350) as illustrated in A. Human M21 melanoma cells were transfected with these PAK4 mutants and compared with cells transfected with wild-type (wt) Flag–PAK4 and a vector containing a nonrelated Flag-tagged BAP protein. Under normal culture conditions, wt PAK4 mainly localized in the cytosol ( B). However, upon cell replating onto VN, the majority of cells transfected with PAK4 displayed a relocalization to lamellipodia ( C). A similar relocalization to lamellipodia upon replating was also observed for the kinase-dead and ΔIBD PAK4 mutants, both of them lacking kinase activity (unpublished data) and PAK4-ΔIBD also lacking integrin-binding capacity ( B). However, the PAK4-L19, 22 that lacks GTPase-binding capacity was found in lamellipodia in almost half of the cells in regular culture and was then redistributed to the membrane in the remaining cells upon replating onto VN. A quantification of the PAK4 relocalization by counting the number of cells with membrane-localized PAK4 is displayed in D. Taken together, these results suggest that PAK4 relocalization to lamellipodia does not require its kinase activity or integrin or Cdc42/Rac binding. However, the Cdc42/Rac binding capacity of PAK4 might be inhibitory for PAK4 localization in lamellipodia.
Figure 5. PAK4 translocation to lamellipodia by integrin ligation to VN does not depend on Cdc42 binding, integrin interaction, or PAK4 kinase activity. (A) Flag-tagged PAK4 mutants used for translocation studies. PAK4-L19, 22 lacks binding capacity to Cdc42/Rac. (more ...)
Dynamic distribution of PAK4 in actively reshaping lamellipodia
To study the temporal and spatial localization of PAK4 in living cells, we established MCF-7 human breast carcinoma cells stably expressing a fluorescent EGFP–PAK4 fusion protein. These cells were plated onto VN-coated glass slides and analyzed by time-lapse fluorescent microscopy. Consistent with our immunofluorescent staining of endogenous PAK4 lamellipodial localization in MCF-7 cells ( A) and of Flag-tagged PAK4 (), EGFP–PAK4 also localized in lamellipodial protrusions after replating onto VN ( A). Interestingly, the PAK4 distribution changed in a highly dynamic fashion, whereas EGFP control cells exhibited only cytoplasmic and nuclear or perinuclear distribution ( B). Furthermore, like endogenous PAK4 ( B), EGFP–PAK4 was also found to partially colocalize with integrin αvβ5 in lamellipodia (unpublished data). The transient localization of PAK4 in lamellipodia coinciding with lamellipodia of actively forming and retracting extensions indicates that PAK4 may modulate these processes. Given that PAK4 associated with integrin αvβ5 and colocalized with integrin αvβ5 in lamellipodia, the dynamic distribution of PAK4 in lamellipodia may reflect a transient and periodic interaction between PAK4 and integrin αvβ5. This led us to hypothesize that PAK4 may not only interact with αvβ5, but that PAK4 may also influence integrin-mediated motile events.
Figure 6. Dynamic localization of PAK4 in lamellipodia. MCF-7 human breast carcinoma cells stably transfected with EGFP–PAK4 or EGFP were plated onto VN and visualized by immunofluorescent microscopy after being allowed to attach for 30 min. Images were (more ...)
PAK4 stimulates integrin αvβ5–specific cell migration in human breast carcinoma cells
Based on the above hypothesis, we examined the potential effect of PAK4 on αvβ5-mediated cell motility. Integrins αvβ3 and αvβ5 both participate in cell attachment and cell migration toward VN (Wayner et al., 1991
). To assess the integrin αvβ5–mediated cell motility, it is ideal to use a cell line expressing αvβ5, but not αvβ3, because αvβ3 usually dominates as VN receptor for cell migration if present, which is the case in most cultured cell lines. Therefore, we chose MCF-7 human breast carcinoma cells, which express αvβ5 but not αvβ3 (unpublished data; Brooks et al., 1997
; Wong et al., 1998
). In addition, we found that both PAK4 mRNA and protein are highly expressed in MCF-7 cells compared with a number of other tumor cell lines tested (unpublished data), consistent with the recent study by Callow et al. (2002)
In a haptotactic cell migration assay, we found that transient expression of EGFP–PAK4 in MCF-7 cells specifically induced MCF-7 cell migration on VN, but not integrin β1–mediated cell migration on collagen type I ( A). Furthermore, EGFP–PAK4-induced cell migration was blocked by a functional blocking anti-αvβ5 mAb, but not by an anti-αvβ3 mAb ( A, left). Taken together, this demonstrates that PAK4 specifically induces integrin αvβ5–mediated cell motility. Moreover, stable expression of EGFP–PAK4 in MCF-7 cells yielded numerically almost identical results on induction of αvβ5-mediated cell migration as transient PAK4 expression, but did not influence cell motility on collagen ( B). Similarly, stable overexpression of a Flag-tagged constitutively active PAK4 mutant (S474E; Callow et al., 2002
) induced MCF-7 cell migration to VN to the same degree as EGFP–PAK4 (unpublished data). This indicates that overexpression of EGFP–PAK4 may saturate PAK4-inducible motility in this cell type, which might be explained by the observation that a large GST fusion partner at the NH2
terminus of PAK1 causes constitutive PAK1 activation, suggesting that the EGFP fusion to PAK4 may cause PAK4 activation.
Figure 7. PAK4 stimulates integrin αvβ5–mediated cell migration. (A) MCF-7 cells transiently transfected with EGFP–PAK4 or EGFP control were analyzed for haptotactic cell migration toward VN in the presence or absence of normal mouse (more ...)
Given that cell adhesion is the basis for cell migration, it was interesting to examine whether PAK4 may also impact cell adhesion on VN. To this end, MCF-7 cells were transfected with EGFP–PAK4 or control EGFP. As shown in C, overexpression of EGFP–PAK4 markedly inhibited cell adhesion on VN compared with EGFP-transfected cells. One possibility that may explain the inhibition of cell adhesion on VN could be a down-regulation of integrin αvβ5 cell surface expression by PAK4. To examine this, we performed a flow cytometry analysis to determine the cell membrane distribution of integrin αvβ5. However, PAK4 overexpression did not change the abundance of integrin αvβ5 expressed on the cell membrane ( D), or that of integrin β1 (unpublished data). This suggests that PAK4 inhibition of cell adhesion might be caused by an alteration of integrin αvβ5 binding capacity for its ligand VN.