Defining the phospho-adhesome through the phosphoproteomic analysis of integrin signalling

Abstract

Cell–extracellular matrix (ECM) adhesion is a fundamental requirement for multicellular existence due to roles in positioning, proliferation and differentiation. Phosphorylation plays a major role in adhesion signalling; however, a full understanding of the phosphorylation events that occur at sites of adhesion is lacking. Here we report a proteomic and phosphoproteomic analysis of adhesion complexes isolated from cells spread on fibronectin. We identify 1,174 proteins, 499 of which are phosphorylated (1,109 phosphorylation sites), including both well-characterized and novel adhesion-regulated phosphorylation events. Immunoblotting suggests that two classes of phosphorylated residues are found at adhesion sites—those induced by adhesion and those constitutively phosphorylated but recruited in response to adhesion. Kinase prediction analysis identifies novel kinases with putative roles in adhesion signalling including CDK1, inhibition of which reduces adhesion complex formation. This phospho-adhesome data set constitutes a valuable resource to improve our understanding of the signalling mechanisms through which cell–ECM interactions control cell behaviour.

LAR protein tyrosine phosphatase regulates focal adhesions through CDK1

“…However, there is increasing evidence from both imaging and proteomic studies to support the hypothesis that CDKs have significant non-nuclear roles. CDK1 has been identified as being present in focal adhesions and recent data indicates that CDK1, along with other CDKs, plays a role in integrin-based cell adhesion and motility (Manes et al, 2003;Robertson et al, 2015). During G2/M transition in vertebrates, phosphorylation of CDK1 Thr 161 is a function of CDK7, a CDK-activating kinase (CAK), and one possibility is that LAR might also regulate CDK7 activity (Desai et al, 1995;Fisher, 2005).…”

“…Proteins localizing to focal adhesions include structural proteins that link integrins to the actin cytoskeleton as well as many other proteins involved in signal transduction. These include the well-documented regulators of focal adhesions, Src and focal adhesion kinase (FAK, also known as PTK2) but also other kinases including members of the cyclin-dependent kinase (CDK) family (Horton et al, 2015;Robertson et al, 2015;Zaidel-Bar et al, 2007). The extracellular domain of LAR is known to interact with components of the extracellular matrix including the lamininnidogen complex and syndecan (Johnson et al, 2006;O'Grady et al, 1998), and imaging and proteomic studies have established that LAR is a component of focal adhesions and can regulate the actin cytoskeleton (Serra-Pages et al, 1995;Zaidel-Bar et al, 2007).…”

The microenvironment controls invadosome plasticity

“…Thus, owing to the dynamic nature of this complex, identifying the full molecular identity of these structures is challenging (Artym et al, 2015;Beaty et al, 2013;Sharma et al, 2013;Valenzuela-Iglesias et al, 2015). Currently, there is no study that is able to define the invadosome proteome as accurately as has been done for focal adhesions (Goicoechea et al, 2014;Robertson et al, 2015). In addition, there are only few studies describing the very existence and role of invadopodia in vivo.…”

Desmosome assembly, homeostasis, and desmosomal disease

“…The evolution of the notion of desmosomes from static, punctuate, adhesive barrier structures to finely regulated multifunctional complexes resulted from the realization that regulatory molecules play a fundamental role in the homeostasis of cell adhesion structures, [58][59][60] including desmosomes. 61 An insight into the mechanisms that regulate the dynamics of keratinocyte adhesion, therefore, requires not only an understanding of desmosomal structure and function but also an understanding of the regulation of its component parts and their responses to perturbations.…”