Probing phosphorylation-mediated protein partnerships

The cerebral cavernous malformation (CCM) multiprotein complex cell-autonomously regulates the stability of endothelial and epithelial cell–cell junctions, and is therefore important in vascular development. KRIT1 (also known as CCM1) and CCM2 are thought to form the core of this protein complex, but to gain further insight into the mechanism(s) and regulation of complex assembly, Kim et al. attached a tandem affinity purification (TAP) tag to KRIT1/CCM1 to isolate associated proteins. They confirmed previously reported interactions of KRIT1/CCM1 with CCM2, ICAP1α (an integrin-binding protein), and heart of glass (HEG, a transmembrane receptor), before analysing phosphorylation sites within the complex. The only high-stoichiometry phosphorylation site in KRIT1/CCM1 was Ser22, which can be targeted by protein kinase A or casein kinase I. Two lower-stoichiometry phosphorylation sites — at Thr151 and Tyr252 — were also found. Tyr252, which has been implicated in

CCM2 binding and nuclear retention of KRIT1/CCM1, is a putative target of Janus kinase 2. The authors postulate that the phosphorylation of multiple Ser/Thr sites, which they identified throughout CCM2, might regulate the interaction of this core complex component with other proteins. Phosphorylation of Ser164, Ser166 or Ser168 within the phosphotyrosine-binding domain domain might, for example, influence the binding of CCM2 to KRIT1/CCM1. Several phosphorylation sites were identified in the amino-terminal Ser/Thr-rich domain of ICAP1. Phosphorylation of Thr38, which is known to increase the affinity of ICAP1 for integrin β1 and thereby suppress integrin activation by talin, was not detected in this study; however, this could reflect an increased association of ICAP1 with KRIT1/CCM1 in the absence of Thr38 phosphorylation potentially favored by the TAP protocol. No phosphorylation sites were detected in HEG1.

The data associated with this publication can be accessed and manipulated through the Cell Migration Gateway, here http://www.cellmigration.org/resource/proteomics/data/phospho_index.shtml

• Kim J, Sherman NE, Fox JW, Ginsberg MH. Phosphorylation sites in the cerebral cavernous malformations complex. J Cell Sci. 2011 Dec 1;124(Pt 23):3929-32 PubMed

Retinal angiogenesis: fibronectin performs a two-fold function

During the development of the retinal vascular system, astrocytes guide the leading tips of endothelial cells by depositing a provisional extracellular matrix (ECM). The precise involvement of the major ECM component fibronectin in retinal angiogenesis has so far not been addressed. Stenzel et al. have now shown that fibronectin is expressed and deposited by astrocytes ahead of the growing retinal vasculature in mice, and have taken a genetic approach to elucidate its function. Using Cre-lox-mediated recombination, the authors specifically ablated fibronectin expression in astrocytes ahead of the growing vasculature, causing a decrease in the radial expansion of the vascular plexus through reduced tip-cell migration. Surprisingly, although endothelial cells express both integrin α5 and integrin β1, which mediate binding to fibronectin, endothelium-specific ablation of integrin α5 caused only a marginal reduction in radial migration, suggesting that endothelial α5β1 is not necessary for outgrowth; instead, it supports the alignment and adhesion of filopodia to the astrocytic scaffold. Binding of vascular endothelial growth factor (VEGF) to fibronectin was recently reported to be required for endothelial cell migration, and Stenzel et al. observed that the absence of fibronectin reduced VEGF receptor (VEGFR)2-mediated signaling through phosphoinositide 3-kinase and Akt. They also showed that the blocking peptide FnIII_13–14, which inhibits VEGF–fibronectin binding, inhibited endothelial cell migration in cell culture and significantly reduced the radial expansion of retinal vessels when injected intraocularly, coincident with a decrease in phosphorylation of VEGFR2 and Akt. VEGF also interacts with heparan-sulfate proteoglycans, the expression pattern of which resembles that of fibronectin on astrocytes, and the authors showed, using genetic deletion, that both of these ECM components synergize to promote the directional migration of endothelial tip cells. So fibronectin carries out both integrin-binding and growth-factor-binding functions during retinal angiogenesis.

• Stenzel D, Lundkvist A, Sauvaget D, Busse M, Graupera M, van der Flier A, Wijelath ES, Murray J, Sobel M, Costell M, Takahashi S, Fässler R, Yamaguchi Y, Gutmann DH, Hynes RO, Gerhardt H. Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis. Development. 2011 Oct;138(20):4451-63. PubMed