In this work we describe a novel interaction between palladin, SPIN90 and Src. SPIN90 (also known as DIP, mDia interacting protein) contains an SH3 domain, three proline-rich regions, a serine/threonine-rich region and a long C-terminus and shows high sequence similarity to WISH [18
]. SPIN90 interacts with several actin-regulating proteins including Nck, N-WASP, βPIX, the Arp2/3 complex, PSD-95 and mDia [19
]. It plays a role in myofibril and sarcomere assembly and depletion of SPIN90 in cardiomyocytes results in sarcomere disruption. Furthermore, it participates in the regulation of filopodia and dendritic spines in neuronal cells [34
] and in actin-based motility by promoting the Arp2/3 activity. Finally, SPIN90 can be modulated by ERK1, a kinase activated by cell adhesion or PDGF[21
Previous reports on palladin and SPIN90 suggest that both proteins act as potent cytoskeletal scaffolds bringing together proteins with different functional modalities into higher-order molecular complexes, whose localization and composition can vary in different cell types and situations. Their role as scaffolds is supported by several observations: (1) the proteins do not have intrinsic enzymatic activity, (2) they contain several domain structures that serve as interaction platforms for actin-associated proteins, (3) the proteins serve as substrates for cytoskeleton modulating kinases, (4) they localize at sites where active actin remodeling takes place, such as lamellipodia and membrane ruffles, and (5) their role in actin remodeling has been demonstrated in knock-down experiments[16
]. The present study establishes a molecular link between palladin and SPIN90 but also demonstrates differences in their ability to regulate actin cytoskeleton.
The interaction is mediated by the poly-proline sequences of palladin and the SH3 domain of SPIN90. Palladin differs from the two other protein family members, myotilin and myopalladin, in that its amino-terminus contains proline-rich stretches, which serve as ligands for SH3, EVH and WW domains and also for profilin[26
]. One of these proline sequences is conserved between myopalladin and the palladin isoform, 4Ig, and this particular sequence has been shown to mediate direct binding between nebulin and myopalladin[27
] and the SH3 domain of LASP-1 and palladin 4Ig[12
]. The LASP-1 binding motif is not contained in the major shorter palladin isoform, 3Ig, whose N-terminal poly-prolines bind to the first SH3 domain of ArgBP2 [14
], to the EVH domain of VASP [13
] and to profilin II[28
]. The amino-terminus also binds another SH3 domain protein, Eps8, but this interaction is not mediated by the SH3 domain[16
]. While the interactions between SH3 domains and poly-proline sequences are specific, they are usually of low affinity, suggesting that they can be rather short-lived and dynamically regulated by both intra- and extracellular cues. This could also be the case between palladin and SPIN90 because in non-muscle cells the proteins co-localize and thus probably interact in actively remodeled actin rich structures such as lamellipodia.
Our results further demonstrate a bidirectional link between palladin and Src. Src activation results in phosphorylation of palladin in cells and, on the other hand, palladin is involved in subcellular targeting of Src. Src family kinases regulate three main cellular functions that ultimately control the behavior of transformed cells: adhesion, invasion and motility, all of which require active remodeling of the actin cytoskeleton[1
]. Src has been previously shown to have several focal adhesion and cytoskeletal substrates, including FAK, vinculin, paxillin, cortactin and ezrin[2
]. Activation of Src induces rapid reorganization of the actin cytoskeleton and formation of structures such as lamellipodia and podosomes. Src can be activated by several mechanisms including ligation of receptor tyrosine kinases such as PDGFR and EGFR. When Src is activated by PDGF treatment it rapidly redistributes from central perinuclear location to the plasma membrane. This process requires intact stress fibers and is mediated by the SH3 domain of Src[30
]. Our results show that knock-down of palladin results in disruption of stress fibers and subsequent inhibition of the relocalization of active Src and SPIN90. Since the formation of membrane ruffles and lamellipodia requires Src activity beneath the plasma membrane, targeting of Src by palladin may be a mechanism to control Src-dependent events.
Similarly to palladin, SPIN90 links directly to Src signaling. Meng et al. showed that SPIN-90 (DIP) is a Src substrate and is involved in targeting effector proteins such as Vav2 and p190RhoGAP [32
]. SPIN90 binds to both p190RhoGAP and Vav2 and targets them to the plasma membrane, where all the three components of this complex are phosphorylated by Src. Inhibition of SPIN90 by dominant-negative mutants or siRNA prevented EGF induced actin organization, redistribution of p190RhoGAP and Vav2 and cell movement. SPIN90 inactivates Rho and activates Rac via p190RhoGAP and Vav2, respectively, in a Src dependent manner. Inactivation of Rho leads to loss of stress fibers and activation of Rac to formation of lamellipodia. Our results show that the knock-down of SPIN90 does not affect the targeting of palladin and Src, in line with the results of Meng et al, who showed that SPIN90 (DIP) did not target Src to the plasma membrane [32
]. Together these results offer a basis for a model where Src and SPIN90 translocate after stimulation to the membrane via a palladin/stress fiber dependent manner. At the membrane active Src phosphorylates p190RhoGAP and Vav2 via SPIN90 leading to loss of the stress fibers and subsequently formation of lamellipodia and cell movement. Since palladin and SPIN90 have been shown to have several interaction partners localizing to membrane ruffles and lamellipodia it is possible that the interactions can also vary during different stages of lamellipodia organization. In a recent study by Eisenmann et al SPIN90 induced non-apoptotic membrane blebbing, which is known to be associated with amoeboid cell movement and thus cancer-cell migration [25
]. This novel finding offers additional support for the assumption that SPIN90 plays an important role in organizing the membrane associated cytoskeleton and cellular movement. Furthermore, fibroblasts derived from palladin negative murine embryos have been to shown to have a greatly diminished motility [17
Previously, palladin has been shown to be phosphorylated both on tyrosine and serine/threonine residues but kinases involved in this have not been identified[10
]. Interestingly, PDGF treatment of A7r5 smooth muscle cells leads to reduced migration of palladin in SDS-PAGE suggesting post-translational modification, possibly phosphorylation of the protein, but this preliminary finding needs still to be experimentally verified[16
]. We extend these findings by showing that palladin becomes tyrosine phosphorylated in cells expressing active Src. It remains to be shown, whether Src directly phosphorylates palladin, or whether a kinase downstream of Src would be the actual effector. One of such potential kinases could be FAK.
The role of tyrosine phosphorylation of cytoskeletal proteins is still mostly unclear. One of the best characterized cytoskeletal substrates of Src is cortactin. Cortactin phosphorylation serves a variety of functions, one of which is to provide binding sites for specific signaling proteins with SH2 domains, e.g. Src family kinases and Nck[33
]. Tyrosine phosphorylation may also induce other types of novel interactions as shown between ezrin and a kelch-repeat protein family member, KTBDB2[4
]. The effects of Src can also be mediated indirectly by other effector proteins, for example by the MAPK family members such as ERK. Src has been shown to directly activate ERK, which is of particular interest since SPIN90 is a substrate of ERK[21
]. Phosphorylation of SPIN90 by ERK1 promotes the interaction between SPIN90-βPIX-WASP complex and the adaptor protein Nck[21
]. These findings offer some clues for the consequences of palladin phosphorylation, but further experiments are needed to elucidate its specific function.
In conclusion our results show that palladin interacts directly with both SPIN90 and Src and that palladin plays a role in maintaining stress fiber integrity and targeting of SPIN90 and Src to sites where active actin remodeling takes place. Thus palladin can act as a true protein scaffold bringing together effector proteins and their cyroskeletal targets.