Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

SKAP-HOM

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_629

Synonyms

Historical Background

SKAP-HOM was originally described in 1998 as a protein homologous to the cytosolic adaptor protein SKAP55 (Marie-Cardine et al. 1998). Using the recombinant SH2 domain of the  Src protein tyrosine kinase Fyn expressed as a GST fusion protein, SKAP55 and SKAP-HOM were purified from lysates of pervanadate-treated human T cells. Furthermore, it was demonstrated that SKAP-HOM acts as a substrate for the Src kinase Fyn and that it binds the adaptor molecule FYB/SLAP/ ADAP. At the same time, Liu et al. reported the identification and the molecular cloning of a protein termed SKAP55-R. Using the yeast two-hybrid system with FYB/SLAP/ADAP as bait, the SKAP55-related protein was identified (Liu et al. 1998). The murine homologue of human SKAP55-R was described in 2000. It was reported that murine SKAP55-R plays a role in IL-6-induced myeloid cell differentiation and that overexpression of SKAP55-R inhibits growth of FDC-P1 myeloid cell line and primary hematopoietic progenitors. Independently, a retinoic acid-inducible protein was described, which is expressed during neuronal differentiation of mouse embryonal carcinoma cells. The protein, termed RA70, is highly homologous to SKAP55 and functions as an adaptor protein of the Src family kinases Fyn, Hck, and Lyn (Kouroku et al. 1998). Five years later, a novel adaptor protein, Pyk2/RAFTK-associated protein (PRAP), was identified using the yeast two-hybrid cloning system. PRAP specifically binds to Pyk2/RAFTK, thereby inhibiting Pyk2/RAFTK kinase activity. In this way, tyrosine phosphorylation of α-synuclein, a presynaptic protein that is involved in the pathogenesis of several neurodegenerative diseases, is inhibited. Furthermore, PRAP was found to function as a substrate for Src family kinases such as Fyn (Takahashi et al. 2003).

Consistently, all researchers reported that unlike SKAP55, which is exclusively expressed in T cells, SKAP-HOM expression is ubiquitous.

Structural Organization

The cDNA of human SKAP-HOM encodes a 359 amino acid polypeptide which possesses a pleckstrin homology (PH) domain followed by a central region containing several tyrosine phosphorylation sites and a C-terminal SH3 domain. The tyrosine motif YEEL could serve as a putative binding site for the SH2 domain of Fyn. A second tyrosine motif YAN might represent a binding site for the SH2 domain of Grb2. SKAP-HOM shows the same overall structure as SKAP55 except for an unique N-terminal coiled-coil domain, which is not present in SKAP55 (Fig. 1). The proteins show an overall 44% homology with the greatest conservation in the PH and the SH3 domains (Liu et al. 1998; Marie-Cardine et al. 1998). Given the size and the sequence similarity, it was hypothesized that SKAP55 and SKAP-HOM might originate from gene duplication. The chromosomal localization is chromosome 7 for human and chromosome 6 for the mouse gene, respectively. The identity in the amino acid sequence between mouse and human proteins is 91%.
SKAP-HOM, Fig. 1

Structure of SKAP-HOM and binding partners

Detailed structural and biochemical analysis including crystallization of the SKAP-HOM N-terminus revealed an unusual four-helix bundle dimerization domain. This dimerization domain mediates self-association and homodimerization in vivo. The isolated PH domain binds to phospholipids in vitro. The potential for allosteric inhibitory interactions between the dimerization domain and the PH domain suggests that SKAP-HOM exists in two states: a closed, docked state and an open free conformation capable of phosphoinositide binding. It was hypothesized that this phosphoinositide-responsive molecular switch regulates subcellular targeting, thereby controlling SKAP-HOM function (Swanson et al. 2008).

SKAP55 binds to ADAP with high stoichiometry. In 2005, Huang and coworkers published the unexpected finding that ADAP-deficient Jurkat T cells also lack SKAP55 expression. It was shown that in the absence of ADAP, SKAP55 is rapidly proteolyzed and the half-life of the protein is decreased (Huang et al. 2005). This finding was further extended in ADAP-deficient mouse T cells where it was shown that both SKAP55 and SKAP-HOM are undetectable at protein level. In contrast, SKAP-HOM-deficient T cells as well as SKAP55-deficient T cells show no changes in ADAP protein expression (Togni et al. 2005; Wang and Rudd 2008).

SKAP-HOM contains several modular interaction domains including a PH domain, a C-terminal SH3 domain, an N-terminal coiled-coil domain, and multiple tyrosine phosphorylation sites (Y). The potential interaction partners are indicated below.

Binding Partners of SKAP-HOM

ADAP constitutively binds to SKAP55 or SKAP-HOM. The dominant binding occurs via the SKAP-HOM SH3 domain which binds to the proline-rich regions in ADAP (Liu et al. 1998; Marie-Cardine et al. 1998). A second interaction was described to occur between the ADAP helical SH3 domain and a noncanonical RKxxYxxY motif in SKAP55. Preliminary data indicated that this RKxxYxxY motif in SKAP-HOM also binds to the SH3 domain of ADAP (Wang and Rudd 2008).

PRAM-1 (PML-retinoic acid receptor alpha-regulated adaptor molecule-1) shares structural homologies with ADAP and is expressed early during myeloid differentiation and in mature granulocytes. Co-immunoprecipitation studies documented a constitutive association between PRAM-1 and SKAP-HOM. Although the interaction site was not characterized, PRAM-1 contains several proline-rich repeats corresponding to the SH3 recognition site (Moog-Lutz et al. 2001).

Another interaction partner of SKAP-HOM is the serine/threonine kinase   HPK1 (hematopoietic progenitor kinase 1) which constitutively associates with SKAP-HOM in mouse B cells. In addition, as shown for SKAP55 in T cells, SKAP-HOM interacts in B cells with RIAM, a Rap1 effector molecule involved in integrin activation (Königsberger et al. 2010).

In murine macrophages, SKAP-HOM has been reported to interact with SHPS-1 (Src homology 2 domain-containing protein tyrosine phosphatase substrate 1, also known as SIRP alpha), a transmembrane receptor of the immunoglobulin (Ig) superfamily. SHPS-1 functions as a scaffolding protein mediating the assembly of adhesion-regulated multi-protein complexes including SKAP-HOM, ADAP, Pyk2, and the tyrosine phosphatase SHP-1 (Timms et al. 1999).

Pathogenic species of Yersinia harbor a plasmid-encoded type III secretion system that translocates virulence-associated bacterial proteins into host cells. YopH (Yersinia outer protein H) is a highly active protein tyrosine kinase. When translocated into host cells, YopH disrupts focal adhesion complexes, thereby causing detachment of infected cells and abrogation of the phagocytic process. It has been demonstrated that YopH dephosphorylates SKAP-HOM, ADAP, and Cas (Crk-associated substrate) in murine macrophages, thus interfering with adhesion-regulated signal transduction pathways (Fällman et al. 2002). The short peptide sequence of SKAP-HOM (DEYDDPF), located in the linker region between the dimerization domain and the PH domain, is recognized by the N-terminal substrate-binding domain of YopH. Based on this SKAP-HOM sequence, cyclic peptides have been designed to inhibit Yersinia infections.

Function of SKAP-HOM in the Immune System

SKAP-HOM seems to play a role during myelopoiesis. When overexpressed in myeloid cells, SKAP-HOM inhibited proliferation without affecting differentiation (Bourette et al. 2005). Furthermore, there is clear evidence that SKAP-HOM plays a role in adhesion events. In murine macrophages, the scaffolding protein SHPS-1 assembles an adhesion-regulated multi-protein complex including SKAP-HOM, ADAP, Pyk2, and the tyrosine phosphatase SHP-1. Timms et al. reported that SKAP-HOM is tyrosine phosphorylated in response to macrophage adhesion to fibronectin (Timms et al. 1999). Macrophages that lack SHP-1 display hyperphosphorylation and show increased adhesiveness. In addition, Bourette et al. showed that macrophage colony-stimulating factor (M-CSF) induces tyrosine phosphorylation of SKAP-HOM in myeloid cells and its association with other proteins including actin (Bourette et al. 2005). Collectively, these data support the hypothesis that SKAP-HOM is involved in the regulation of actin dynamics in myeloid cells.

In T cells, ADAP and SKAP55 play an essential role in TCR-mediated inside-out signaling that is needed for LFA-1 clustering and conjugate formation between T cells and antigen-presenting cells (APC). Given the high homology, the question arises whether SKAP-HOM plays a redundant role in the same events. According to Wang and coworkers, SKAP-HOM failed to compensate for the loss of SKAP55 in LFA-1 clustering in T cells (Wang and Rudd 2008).

SKAP-HOM-deficient B cells show strongly reduced adhesion to fibronectin and ICAM-1 after BCR stimulation, whereas B lymphocyte development and differentiation were normal (Togni et al. 2005). The signaling module regulating activation of B-cell integrins involves HPK1, SKAP-HOM, and RIAM. The loss of HPK1 induces enhanced integrin activity suggesting a negative regulatory role for this signaling complex. In conclusion, these data indicate that SKAP-HOM and SKAP55 may have similar but specialized functions in T and B cells (Königsberger et al. 2010).

In SKAP-HOM-deficient bone marrow-derived dendritic cells (BMDCs), the spontaneous motility is enhanced. By contrast, adhesion of mature BMDCs to fibronectin is reduced, and antigen-dependent conjugate formation between SKAP-HOM-deficient DCs and T cells is delayed (Reinhold et al. 2009). Bone marrow-derived macrophages from SKAP-HOM-deficient mice exhibit abortive actin-rich ruffle formation (Swanson et al. 2008).

Overall, the results obtained from the characterization of the knockout mice (Table 1) confirm the role of SKAP-HOM in the regulation of integrin activation and adhesion in immune cells.
SKAP-HOM, Table 1

Summary of the characterization of SKAP-HOM-deficient mice

Cell type

Phenotype

References

T cells

Normal

Togni et al. (2005)

B cells

Reduced proliferation

Togni et al. (2005)

Reduced BCR-mediated adhesion to fibronectin and ICAM-1

Reduced serum immunoglobulins

Platelets

Normal spreading and aggregation

Togni et al. (2005)

Normal platelet activation

Bone marrow-derived dendritic cells (BMDCs)

Increased migration into the lymph nodes in vivo

Reinhold et al. (2009)

Higher motility in vitro

Reduced adhesion to fibronectin

Delayed conjugate formation with antigen-specific T cells

Reduced proliferation of antigen-specific T cells

Bone marrow macrophages (BMM)

Normal proliferation

Togni et al. (2005), Swanson et al. (2008)

Normal phagocytosis

Impaired ruffle formation

Disease model

Reduced severity of experimental autoimmune encephalomyelitis (EAE)

Togni et al. (2005)

Function of SKAP-HOM Outside the Immune System

During the last years, knowledge about the role of SKAP-HOM outside the immune system is constantly increasing. For example, heat shock factor HSF4b belongs to the transcriptional regulator family of heat shock proteins (Hsp). HSF4b is mainly expressed in lens epithelial cells and plays a major role in lens development. SKAP-HOM was identified as a novel target of HSF4b that associates with Nck2 and F-actin at membrane ruffles, thereby regulating actin remodeling in lens cells (Zhou et al. 2011).

In an attempt to search for novel genes that are involved in pancreatic carcinogenesis, SKAP-HOM was the most frequently detected overexpressed gene in microdissections of pancreatic ductal adenocarcinoma specimen compared to reference samples. There was a significant correlation between DNA copy number and mRNA expression level. This dysregulation of SKAP-HOM expression might be associated with the development of pancreatic cancer (Harada et al. 2008). Another study was performed to identify progression-associated metastasis genes in malignant melanoma. Functional assays were performed on a subset of 30 candidate genes including SKAP-HOM. siRNA-mediated knockdown of SKAP-HOM significantly inhibited invasion of melanoma cells in the Boyden chamber assay (Kabbarah et al. 2010). Tumor-associated macrophages can support tumor cell growth and metastasis and are associated with poor prognosis. SKAP-HOM is highly expressed in macrophages in various tumor tissues. In vitro studies showed that SKAP-HOM is essential in tumor-infiltrating macrophages for podosome formation and matrix degradation. In vivo, SKAP-HOM-deficient macrophages could not efficiently accumulate in mouse lung tumors, thus reducing lung metastasis (Tanaka et al. 2016). These data indicate that high expression of SKAP-HOM in invasive tumor cells as well as in tumor-infiltrating macrophages promotes tumor invasion and metastasis.

The genetic association of HLA class II genes and susceptibility to type 1 diabetes is well established. Genome-wide association studies found over 40 non-HLA-risk loci. One of the identified risk loci is located on chromosome 7 (7p15.2) and contains the SKAP2 gene. A single nucleotide polymorphism, located within the SKAP2 gene, was found to be associated with the risk of type 1 diabetes. Recent functional studies revealed that this single nucleotide polymorphism significantly downregulated the expression of the SKAP2 gene. This observation highlights a possible role of SKAP-HOM in the development of autoimmunity. SKAP-HOM is a type 1 diabetes candidate gene of special interest because it is expressed within the human islets as well as in immune cells (Storling and Brorsson 2013).

SKAP-HOM expression is also found in murine brain structures as the hippocampus, frontal cortex, and cerebellum. Behavioral testing of SKAP-HOM-deficient mice showed impaired social memory and accelerated extinction of conditioned fear, whereas hippocampus-dependent learning in general was not affected. It was hypothesized that the signal transduction adaptor protein SKAP-HOM might be involved in complex interactions between the immune system and cognitive functions (Fiedler et al. 2014).

Summary

SKAP-HOM is a ubiquitously expressed cytosolic adaptor molecule. In immune cells, the molecular complex formed between SKAP-HOM, ADAP, and RIAM is involved in the regulation of inside-out signaling from immunoreceptors to integrins. To dissect the specialized function of SKAP-HOM and SKAP55 in T cells, an analysis of the respective knockout mice in parallel with a SKAP-HOM/SKAP55 double knockout mouse would be helpful. Further studies are needed to investigate the function of SKAP-HOM outside the immune system. The potential role of SKAP-HOM as a cancer-related gene seems to be of special interest. The knowledge of SKAP-HOM interaction partners and their protein-protein interaction domains might provide useful information for the design of synthetic inhibitors to modulate the invasive phenotype of tumor cells.

References

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Institute for Molecular and Clinical ImmunologyOtto von Guericke University MagdeburgMagdeburgGermany