The parafibromin tumor suppressor protein interacts with actin-binding proteins actinin-2 and actinin-3

Parafibromin is encoded by the HRPT2 gene located on chromosome 1q31[1] (OMIM# 145001 and 607393). HRPT2 inactivating mutations in the germline occur in the hyperparathyroidism-jaw tumor (HPT-JT) syndrome, characterized by hyperparathyroidism (parathyroid adenoma or carcinoma) (90%), fibrous-ossifying jaw tumors (30%), and bilateral renal cysts (10%) [1, 2]. Germline HRPT2 mutations have also been detected in some cases of familial hyperparathyroidism [1, 3, 4], and biallelic somatic inactivation of HRPT2 is common in parathyroid tumor of HPT-JT and in sporadic parathyroid cancer [5‐7].

Parafibromin has three possible nuclear localization signals; it is predominantly found in the nucleus [8‐16] and in the nucleolus [17]. The C-terminal part of parafibromin (ORF = 531 amino acids) is homologous to yeast Cdc73p (ORF = 393 amino acids), an RNA polymerase II accessory protein [18]. Analogous to the presence of yeast Cdc73p in the yeast Paf1 protein complex, parafibromin was found in the human and Drosophila PAF1 protein complex [10, 19‐21]. Genetic interaction studies in Drosophila and protein interaction studies in mammalian cells have shown an interaction of parafibromin with β-catenin, mediating a functional association between Wnt signaling and the PAF1 complex [22].

The yeast two-hybrid system has been extensively used for the identification and analysis of protein-protein interactions [23, 24]. To isolate parafibromin interacting proteins we conducted a yeast two-hybrid library screen, and we discovered an interaction between parafibromin and muscle actinins.

One of the functional associations of parafibromin is with the PAF1 complex, involved in the regulation of transcriptional initiation and elongation in yeast, and based upon the presence of a C-terminal domain homologous to yeast Cdc73p [10, 19‐21]. This function of parafibromin is also connected to the Wnt signaling pathway by interaction with β-catenin [22]. In this interaction, parafibromin possibly recruits the PAF1 complex to β-catenin responsive genes thereby modulating the transcriptional output of β-catenin. In reporter assays, β-catenin mediated transcription could be regulated by Drosophila parafibromin (hyrax) but not by yeast Cdc73p [22]. In Drosophila with mutant hyrax alleles, human or Drosophila parafibromin could rescue the lethal phenotype [22]. The lethal phenotype of mutant hyrax alleles was not rescued by yeast Cdc73p. The presence of the N-terminal 350 amino acid region in human and Drosophila parafibromin that is not found in yeast Cdc73p suggests that, in addition to its Cdc73-associated function, parafibromin could possess other activities and/or functional associations.

Using the yeast two-hybrid approach, we report interaction of the N-terminus of parafibromin with muscle actinins. Multiple (n = 10) independent clones of actinin-2 were isolated in the yeast two-hybrid library screen, and the interaction was verified by other in vitro and in vivo methods suggesting a strong and significant interaction between parafibromin and actinin-2. Actinins (molecular mass ~100 kDa) are actin-binding proteins that are expressed in both muscle and non-muscle cells, and that cause bundling of actin filaments for cytoskeletal organization [29]. Actinins also serve to link the cytoskeleton, membrane proteins, and signaling proteins [30, 31]. There are 4 alpha-actinins encoded by 4 different genes. The 4 proteins are highly homologous sharing 73–84% amino acid sequence identity [32]. Actinin-1 and actinin-4 are abundant in non-muscle cells, forming actin filament bundles such as stress fibers. They are associated with calcium dependent membrane attachment, and with cell-cell and cell-matrix adherent junctions [33]. In muscle cells, actinin-2 and actinin-3 are major structural components of striated muscle where they anchor actin filaments [34]. Actinin-2 is expressed in all muscle cells, whereas actinin-3 expression is limited to a subset of fast twitch skeletal fibers. In our yeast two-hybrid assays, parafibromin selectively interacted with the two muscle actinins (actinin-2 and actinin-3) but not at all with the non-muscle actinins (Table 2).

Actinins are composed of three distinct domains. An N-terminal actin-binding domain, a central rod-shaped domain with 4 internal spectrin-like repeats required for anti-parallel dimerization, and a C-terminal calmodulin-like domain containing a pair of calcium-binding EF hands [35] (Figure 2B). The EF-hands in non-muscle actinins, actinin-1 and actinin-4, are functional (calcium sensitive) whereas the EF-hands in muscle actinins, actinin-2 and -3 are non-functional (calcium insensitive). All the library clones of actinin-2 interacting with parafibromin were truncated at the N-terminus thus lacking the actin-binding domain and most of the first spectrin repeat of actinins. Further deletion of the N-terminus including the second spectrin repeat prevented interaction with parafibromin. Similarly, actinin-3 lacking its C-terminal EF-hand domains allowed interaction with parafibromin, but further C-terminal deletion including the third and fourth spectrin repeat (but retaining the first and second spectrin repeat) impaired interaction with parafibromin. Therefore, parafibromin likely interacts with the region of actinin containing spectrin repeats 2, 3 and 4 (Figure 2B). The region of highest variability among all actinins is in spectrin repeats 3 and 4 (not shown in Figure 2B) [36]. This could potentially explain why parafibromin interacts with muscle actinins and not with non-muscle actinins. Deletion of N-terminal 112 amino acids of parafibromin impaired actinin interaction. The N-terminal region of parafibromin minus its C-terminal Cdc73p homologous domain was sufficient for interaction with actinin (Figure 2A).

Actinins possess actin bundling and cross-linking activity [26, 27, 35]. In low speed actin sedimentation assays where actin filaments (F-actin) pellet only when they are arranged in higher order structures by cross-linking and/or bundling, just like actinin, to our surprise parafibromin was also capable of pelleting F-actin, indicating that parafibromin probably has inherent F-actin bundling or cross-linking activity. Moreover, despite parafibromin's predicted interaction with a part of the actinin dimerization region, parafibromin did not displace or disrupt actinin from F-actin bundles based on the observation that presence of parafibromin and actinin together appeared to slightly enhance the pelleting of F-actin (Figure 3B). This assay does not distinguish if the increased F-actin in the pellet fraction upon incubation of F-actin with actinin and parafibromin together was due to an increase in the number of F-actin bundles, or due to thicker bundles resulting from an increase in the number of F-actin filaments in individual bundles, or due to increased cross-linking. In yeast two-hybrid assays parafibromin did not interact with actin (Table 2). Parafibromin sequence analysis did not reveal any regions with significant homology to the several different actin-binding motifs that are found in actin-binding proteins [37].

Skeletal muscle myoblasts (such as modeled by C2C12 cells) can be induced to differentiate in mitogen-poor media to fuse and form multinucleate myotubes. This myogenic program is coupled to loss of cell proliferation and increase of differentiation. Actinins are important cytoskeletal structural proteins and their expression in the multinucleate myotubes increases upon induction of myotube formation [28]. Parafibromin's accumulation in the cytoplasm coincides with the inhibition of proliferation and with the activation of differentiation. The co-localization of parafibromin and actinin in the cytoplasm could be unrelated to their direct interaction. But the changed compartmentalization in HEK293 cells of endogenous parafibromin to the cytoplasm upon actinin transfection (Figure 6) indicates that the observation in C2C12 cells is likely related to parafibromin-actinin interaction. Parafibromin-actinin interaction might be directly important for mechanical strength and structure of the actin cytoskeleton, or the interaction could mediate other regulatory functions. Actin and actinin could play a role in regulating parafibromin levels in the nucleus for modulating parafibromin's transcription associated functions. The transcription associated tumor suppressor function of parafibromin could require its migration from the nucleus to cytoplasm as in C2C12 cells. Actin has been implicated in regulating gene transcription through several mechanisms, one of which is a direct role in controlling the nucleo-cytoplasmic distribution of transcription factors (Reviewed in [38]). Therefore, this interaction could be important for regulating the activity, and sub-cellular localization of parafibromin. Our observations in C2C12 cells also suggest other new aspects of parafibromin action such as a possible role in organization of the cytoskeleton and in myotube differentiation. We have investigated parafibromin-actinin interaction in muscle cells. The functional consequence of parafibromin-actinin interaction in cells affected in HPT-JT – parathyroid cells, bone cells, or renal cells – remains to be determined.

In C. elegans, RNAi against HRPT2 caused defects in the proper formation of polar bodies, pronuclei and mitotic nuclei indicating that HRPT2 is important for the proper completion of both meiosis and mitosis [39]. The phenotype analysis also suggested that the HRPT2 product is important for microtubule-based function in the early embryo. Perhaps, this function of parafibromin is also linked to its association with actinins.

Our study does not directly address parafibromin's participation in the PAF-1 complex or the physiological function of parafibromin in the nucleus or cytoplasm, but the observation that interaction with actinins (and actin filament bundling/cross-linking) likely occurs when parafibromin accumulates in the cytoplasm suggests that a biological activity of parafibromin might be controlled by signals that promote its cytoplasmic accumulation. Further studies to recognize such signals and their consequences could provide insight into the functions of parafibromin in normal physiology and in tumorigenesis.