A potential mitotic function of src proteins has been the subject of speculation for more than a decade. However, direct analysis of this potential cell cycle function has been awaiting the identification of a mitotic substrate of src kinases. The first such substrate was Sam68, an RNA binding protein whose RNA binding activity is inhibited by tyrosine phosphorylation (Taylor and Shalloway 1994
; Fumagalli et al. 1994
). The role of this phosphorylation in regulating mitotic events remains to be defined. We present here a second mitotic substrate of src kinases, a transmembrane protein that functions in cell adhesion. The cell cycle and adhesion functions of this novel src substrate suggest the new hypothesis that src may regulate the mitotic changes in cell adhesion. Cell adhesion is generally presumed to be regulated during mitosis, particularly in epithelial cells which need to release themselves from their cell-matrix and cell-cell interactions transiently in order to allow cell duplication and separation of daughter cells. However the molecular mechanism that regulates this dynamic cell adhesion process is unknown. Using an inducible overexpression model, we show that Trask functions in cell adhesion. Trask is unique among currently described cell adhesion molecules in that it is under cell cycle regulation and is a candidate protein for regulating cell adhesion during mitosis.
The adhesion functions of Trask could be regulated through its phosphorylation. In our inducible Trask overexpression model, the induced Trask, unlike endogenous Trask, undergoes significant interphase tyrosine phosphorylation while producing a loss of adhesion phenotype. The excessive interphase phosphorylation of the induced construct may be due to the excessive nature of its expression in this model system. Whether the phosphorylation of Trask is the cause or the consequence of the loss of adhesion remains to be determined. Phosphorylation of Trask could mediate structural changes in its conformation leading to altered interaction of its extracellular domain, or could promote or inhibit its interaction with other transmembrane proteins involved in cell adhesion. Phosphorylation could also recruit cytoplasmic signaling proteins, in particular SH2 containing proteins involved in adhesion signaling. These are potential mechanisms by which Trask phosphorylation could influence interactions with other cell adhesion proteins and signal changes in cell adhesion.
The mitotic phosphorylation of Trask also potentially implicates src kinases in the regulation of Trask function and ultimately in the mitotic regulation of cell adhesion. In addition to MDA-468 cells, the mitotic hyperphosphorylation of Trask is evident in a number of other epithelial tumor cell lines with activated src kinases, but not readily apparent in an immortalized breast epithelial cell line or in fibroblasts (not shown).
In addition to its phosphorylation, the expression and localization of Trask are also regulated during the cell cycle. While the Trask RNA remains low during most of interphase with a small increase during S-phase, Trask protein expression increases during S, G2 and M phases. This is likely due to changes in mRNA stability and other post-transcriptional or post-translational mechanisms of regulation. Taken together, there is clear evidence of the cell cycle regulation of Trask at the level of expression, localization and post-translational modification by phosphorylation.
Trask function and expression may be important in human tumor progression. Consistent with this, Trask has also been identified by two other groups using global strategies to identify tumor-associated genes. Using cDNA chip hybridization techniques to search for genes preferentially expressed in solid tumors relative to normal tissues, Mostageer et al identified Est sequences corresponding to a cDNA identical to Trask, named CUB domain containing protein 1 (CDCP1), although the protein product was not identified in this study (Scherl-Mostageer et al. 2001
). Of note, these authors use algorithms that predict a third CUB domain in the extracellular region of Trask, however in our analysis we find only two CUB domains and with low homology scores. The proposed third CUB domain falls below the threshold of homology of existing public domain algorithms for protein domain prediction. In addition, CUB domains are often only loosely related at the primary structural level making sequence-based predictions difficult. The ultimate characterization of the nature of these domains in Trask thus awaits tertiary structural analysis. In another approach to identify tumor-associated proteins, Hooper et al used subtractive immunization techniques to generate antibodies towards cell surface epitopes preferentially expressed by highly metastatic relative to non-metastatic carcinomas, and identified a cell surface glycoprotein named SIMA135, identical to Trask, and their analysis identifies two extracellular CUB domains (Hooper et al. 2003
). We have independently cloned Trask as a cell cycle substrate of src kinases with a specific adhesion phenotype and interactions with adhesion proteins. We describe here the full length protein, its cell cycle expression pattern and its mitotic relocalization, its specific cleavage at Arg369
by MT-SP1, its stable interaction and specific cell cycle phosphorylation by src and yes, and its interaction with matrix and adhesion proteins and with MT-SP1. Trask is the first adhesion molecule described that is under cell cycle regulation and its association and mitotic phosphorylation by src kinases, for the first time, implicates src in the regulation of adhesion in epithelial cells undergoing mitosis.
Proteolytic processing of Trask may a mechanism of regulation of its function. Trask is expressed predominantly in its cleaved 85kd form in MDA-468 breast cancer cells and we see no shifting between cleaved and uncleaved forms during cell cycle progression in these cells (data not shown). However there are substantial differences in the expression of cleaved and uncleaved Trask among various cancer cell lines, with predominant expression of cleaved Trask in most (not shown). Consistent with this, MDA-468 breast cancer cells, like many other epithelial cancer cell lines, exhibit characteristic upregulation of extracellular proteolytic pathways including activated MT-SP1. MT-SP1, which interacts with and cleaves Trask, is upregulated in many common human cancers including breast, ovarian, prostate, and colon cancers and may be responsible for Trask cleavage in most cancer cells (Bhatt et al. 2003
; Santin et al. 2004
). Additional serine proteases may also be able to cleave Trask. The addition of exogenous plasmin to human keratinocytes results in cleavage of gp140 to p80 and this protein is felt to be CDCP1/Trask based on anti-Fak immunoblots that seemingly cross react with the CDCP1/Trask protein (Brown et al. 2004
) Although the direct significance of Trask cleavage has not yet been ascertained, it holds promise as an interesting mechanism of regulation of Trask activity.
The interaction of Trask with MT-SP1 may have specific functional implications. MT-SP1 has two extracellular CUB domains that may provide the structural basis for the interaction with Trask, since Trask also has CUB domains and CUB domains are known to be involved in homophilic interactions. MT-SP1 cleaves Trask and releases the N-terminal CUB domain. This would result in the release of binding partners of Trask that interact specifically with the N-terminal CUB domain. This may include adhesion molecules, extracellular peptide growth factors, soluble proteases, extracellular matrix components, matrix metalloproteases, or components of the plasminogen activation cascade. In addition to being a substrate of MT-SP1, Trask may play a role in regulating MT-SP1 activity or mediating the interaction of MT-SP1 with membrane and matrix proteins or with components of proteolytic cascades. MT-SP1 cleaves a number of cancer-associated proteins. In particular, MT-SP1 cleaves and activates urokinase-type plasminogen activator (uPA) which can trigger the plasminogen activation cascade (Takeuchi et al. 2000
). Trask may provide a docking surface for MT-SP1 to interact with and activate uPA, leading to juxtamembrane activation of the plasminogen cascade, and increased invasiveness of cancer cells. MT-SP1 also cleaves pro-hepatocyte growth factor (pro-HGF) (Lee et al. 2000
). Trask may also facilitate this interaction leading to increased HGF signaling which is associated with increased motility and invasiveness of tumor cells. Similarly, through a potential docking function, Trask may mediate the interaction of MT-SP1 with other soluble proteases involved in matrix remodeling such as matrix metalloproteases.
Trask is clearly a substrate of src family kinases within the cell and its function is likely modulated by phosphorylation of the intracellular domain. Although in MDA-468 cells Trask interacts predominantly with yes, it is difficult to draw any conclusions with regards to role of individual src family members in regulating Trask or mitotic processes, since src, yes, and fyn have largely overlapping functions. Yes, like src, is also activated in human cancers (Pena et al. 1995
; Park et al. 1993
). In 293 cells Trask interacts equally with both yes and fyn (data not shown). Src kinases are well-established players in oncogenesis and as more specific inhibitors of these kinases become available, a clearer delineation of the specificity and mechanism of Trask phosphorylation will be ascertained.
Trask is only the second mitotic substrate of src kinases identified to date and the first adhesion molecule found to be under cell cycle regulation. We have shown that Trask is phsophorylated in a cell cycle specific manner and that Trask represents a novel class of adhesion-related proteins. The phosphorylation of Trask represents a first possible method of regulation. Proteolytic cleavage of Trask is mediated by an extracellular proteolytic system and this may be yet another method of Trask regulation. This dual control and the identification of an adhesion-related, cell cycle substrate of src kinases presents a new paradigm to study the physiologic role of src kinases in mitosis and the pathologic role of src kinases in the mediating the invasive and metastatic properties of src driven tumors.