Pleckstrin was first initially described as a prominent substrate of protein kinase C (PKC) in hematopoietic cells. In its naming, “PLEC” is derived from platelet and leukocyte C kinase substrate, and “KSTR” is derived from the amino acid sequence KSTR. Pleckstrin protein sequences are highly conserved across human and mouse, which is about 97% homologous to each other. Accordingly, the protein structures and functions of these two different species are also almost same. Pleckstrin-1 (PLEK1), specifically expressed in hematopoietic cells, is composed of two pleckstrin homology (PH) domains at the amino- and carboxyl-terminal and a central disheveled-Egl-10-pleckstrin (DEP) domain (Figure 1A). As a major substrate of PKC in platelets and leukocytes, the phosphorylation of PLEK1 was used as a marker for platelet activation (Hu et al., 1999; Inazu et al., 1999).

Pleckstrin-2 (PLEK2) is another member of pleckstrin family, which is 65% homologous and 39% identical to paralog PLEK1 (Figure 1B). Similar to PLEK1, PLEK2 harbors a central DEP domain flanked by two PH domains (Figure 1A). However, PLEK2 is widely expressed, especially in thymus, stomach, large and small bowels, and prostate (Hu et al., 1999; Inazu et al., 1999).

Both isoforms can induce the formation of large lamellipodia and peripheral ruffle of cells, thereby facilitating cell spreading, in which PLEK1 is entirely regulated by its phosphorylation by PKC (Ma and Abrams, 1999; Roll et al., 2000). In contrast, PLEK2 is not efficiently phosphorylated by PKC but rather by the local generation of PI3K-phosphorylated phospholipids (Bach et al., 2007; Hamaguchi et al., 2007). Recently, roles of PLEK2 in other cellular events beyond cell spreading are gradually being identified. Several lines of evidences suggest that PLEK2 is a potential therapeutic target for the treatment of cancers (Zhao et al., 2018; Shen et al., 2019; Wu et al., 2020; Liu et al., 2021; Wang et al., 2021a; Yang et al., 2021).

Three-dimensional (3D) protein structures have contributed enormously to our understanding of atomic and molecular structure, and help delineate complex macromolecules and their functions, as well as how they operate in the real world. Although the amino sequence of PLEK2 has been characterized with well-defined domains, its protein architecture is poorly investigated. Only the solution structures of C-terminal PH domain and DEP domain of PLEK2 had been reported, respectively (Table 1). Due to the similarity in the structure of the two proteins, the studies on structure of PLEK1 may provide insights for the study of PLEK2. C-terminal PH domain of human PLEK1 (PDB code:1XX0, 1 × 05) was first reported by two independent groups in 2005 (Edlich et al., 2005). Its crystal structure was identified in 2006 (PDB code:1ZM0) (Jackson et al., 2006) and further confirmed by the crystal structure in complex with D-myo-Ins(1,2,3,5,6)P5 (PDB code:2I5F) and D-myo-Ins(1,2,3,4,5)P5 (PDB code:2I5C), respectively, in 2007 (Jackson et al., 2007). Furthermore, it has been suggested that myo-inositol pentakisphosphates regulate the interaction between PH domain and phosphoinositides through direct competition binding to PH domain (Jackson et al., 2007). Although the solution structures of DEP and N-PH domains had been published, their physical structures need to be further confirmed by protein crystal.

The structures of PH domain and DEP domain had been well characterized, respectively, however, full-length structures of PLEK1 and PLEK2 are still largely unknown. Improvements in protein production, crystallization, as well as structure solution and refinement methods have brought the field to the verge of rapid protein structure determination. The major bottle neck to this process remains protein production and crystallization. It has been suggested that DEP interacts intramolecularly with the N-terminal PH domain to mediate membrane anchoring of PLEK1 (Civera et al., 2005). Such interaction may also occur between two PLEK molecules, which accounts for the formation of irregular polymers of PLEK2. This is further supported by the endogenous oligomerization of PLEK2 (Hamaguchi et al., 2007). Indeed, our group previously tried to acquire the protein crystal of PLEK2 but with no success. The failure was due to the difficulty to purify the protein monomer from irregular polymers of PLEK2.

Examining the crude 3D model built with the sequence for mouse PLEK2, two putative small molecule binding grooves appear to exist near K13 and R14 residues (N-PH domain) and at K256 residues (C-PH domain), respectively. Consistently, these sites have been suggested to be response for the phospholipid binding domains (Bach et al., 2007; Jackson et al., 2007). Furthermore, at N156,N157 and D166 (DEP domain) also a potential ligand binding groove exists in the preliminary model.

Due to the highly similarity of protein structural features, it is not surprise that both of these two pleckstrin proteins regulate cell protrusions such as lamellipodia and filopodia, which are important for cellular shape change and spreading (Figure 2). Meanwhile, PLEK2 exhibits distinct roles in other cellular events through the individual molecular interactions and regulatory mechanisms (Table 2).

Interaction partners are essential for the study of molecular mechanism of proteins in cell biology. Our understanding of PLEK2 function is limited mainly due to the poorly identification of interaction partners (Table 2). As mentioned above, PLEK2 interacts with PI (3,4,5) P3 and PI (3,4) P2 generated by PI3K, to anchor on the cell membrane and regulate lamellipodia formation and cell spreading (Bach et al., 2007; Hamaguchi et al., 2007). On the other hand, PLEK2 directly binds to Actin (Hu et al., 1999; Zhao et al., 2014), or interacts with Rac GTPase (Hamaguchi et al., 2007; Feola et al., 2021), to regulate the cytoskeleton dynamics. Similarly, Rac has also been reported to be interplayed with PLEK1 to reorganize the cytoskeleton in addition to integrin (Ma and Abrams, 1999; Roll et al., 2000).

SHIP2, well known to negatively regulate the signaling via dephosphorylation of the 3-position of PI(3,4,5)P3 to generate PI(4,5)P2, is one of the two recently identified interaction partners of PLEK2. PLEK2 mediates degradation of SHIP2 in a ubiquitin-dependent manner, which is further activated PI3K/AKT signaling to promote lung cancer metastasis and vascular invasion (Wu et al., 2020). The other one is EGFR, which is involved in the EMT process activation in gallbladder cancer. PLEK2 interacts with EGFR to protect it from proteasomal mediated degradation, leading to constitutive activation of EGFR signaling (Shen et al., 2019).

As our understanding of the biological functions of PLEK2 increases, it is becoming clear that PLEK2 plays important roles in other cellular events beyond cell spreading, such as inflammation, erythropoiesis, tumorigenesis and metastasis (Figure 3). However, its regulatory roles in these cellular activities remains poorly understood. Further identification of interaction partners and exact regulatory mechanisms that are involved in such cellular process regulation, is required for a full understanding of the functional roles of PLEK2 and the development of targeted therapy.

The role of PLEK2 in tumor development is gradually being recognized, and it serves as a potential diagnostic and prognostic biomarker as well as an attractive target for the treatment of cancers. However, there is no reported small molecule inhibitor for PLEK1 or PLEK2. Therefore, the development of small molecules that modulate the function of PLEK2 is not only beneficial for the treatment of diverse cancers, but also provides extremely useful tools for studying PLEK2 functions as a powerful complementary method to the genetic methods.

Importantly, the three-dimensional protein structure is essential for designing and generating small molecules targeting PLEK2. Although there is no reported full-length structure of PLEK2, the prediction of the 3D structure of PLEK2 based on the solution structures of fragments through homology modeling methods (Vyas et al., 2012; Jaramillo-Martinez et al., 2021), could provide potential small molecule binding pockets for the high-throughput virtual screening and subsequent drug discovery. This promises to enable to validate and use PLEK2 as drug targets in the future.

GW, QZ, and BZ conceived the manuscript. GW and BZ wrote the manuscript. GW and YX contributed to crafting figures. GW, YX, and BZ reviewed and edited the manuscript. All authors listed have made a substantial, direct and intellectual contribution to work, and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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