Integrin cytoplasmic domain‐associated protein‐1 (ICAP‐1) interacts with the ROCK‐I kinase at the plasma membrane

Abstract

The integrin cytoplasmic domain-associated protein-1 (ICAP-1) binds via its C-terminal PTB (phosphotyrosine-binding) domain to the cytoplasmic tails of beta1 but not other integrins. Using the yeast two-hybrid assay, we found that ICAP-1 binds the ROCK-I kinase, an effector of the RhoA GTPase. By coimmunoprecipitation we show that ICAP-1 and ROCK form complexes in cells and that ICAP-1 contains two binding sites for ROCK. In cells transfected with both ICAP-1 and ROCK, the proteins colocalized at the cell membrane predominantly in lamellipodia and membrane ruffles, but also in retraction fibers. ROCK was not found at these sites when ICAP-1 was not co-transfected, indicating that ICAP-1 translocated ROCK. In lamellipodia ICAP-1 and ROCK colocalized with endogenous beta1 integrins and this colocalization was also observed with the isolated ICAP-1 PTB domain. The plasma membrane localization of ROCK did not depend on beta1 integrin ligation or ROCK kinase activity, and in truncated ROCK proteins it required the presence of the ICAP-1-binding domain. To show that the interaction was direct, we measured fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) fused to ICAP-1 and yellow fluorescent protein (YFP) fused to ROCK. FRET was observed in lamellipodia in cells that were induced to spread. These results indicate that ICAP-1-mediated binding of ROCK to beta1 integrin serves to localize the ROCK-I kinase to both the leading edge and the trailing edge where ROCK affects cell migration.

Concepts and hypothesis: integrin cytoplasmic domain-associated protein-1 (ICAP-1) as a potential player in cerebral cavernous malformation

“…As for ICAP-1 cytoplasmic localization, overexpression of b1 integrin sequestered ICAP-1 in the cytoplasm and reduced its nuclear localization [27], consistent with the cytoplasmic localization of ICAP-1, together with its binding partners such as Nm23-H2 [26] or Rho kinase (ROCK) [2,59], at b1 integrin engagement sites. However, it remains unclear what role ICAP-1 and CCM1 might play through their cytoplasmic localization.…”

“…ICAP-1 is a key regulator in b1 integrin signaling [10,11,23,59], and one recent study established its critical role during vessel development [13]. Loss of ICAP-1 function causes excessive microvessel growth, typically reflecting the abnormal vessel development associated with pathogenesis of CCM.…”

ROCK1 Is Associated with Alzheimer’s Disease-Specific Plaques, as well as Enhances Autophagosome Formation But not Autophagic Aβ Clearance

“…The kinases that are involved in phosphorylating Aβ are poorly understood, but are thought to be of the AGC kinase family of serine-threonine kinases (Rezaei-Ghaleh et al, 2016), which includes ROCK1. Unlike ROCK2, ROCK1 interacts with a number of proteins in the plasma membrane (Stroeken et al, 2006) and we show that ROCK1 co-localizes with highly fibrilized Aβ in human brain. The localization and association of ROCK1-associated Aβ with clinical AD mirrors the data observed for serine 8 phosporylated Aβ (Rijal Upadhaya et al, 2014) and suggests that ROCK1 may be the kinase involved in this disease-specific Aβ phosphorylation.…”

Rho-associated coiled-coil containing kinases (ROCK)

“…proteinatlas.org). Several lines of evidence also point to ROCK1 association with the plasma membrane 48,49 and centrosomes. 50 Interestingly, Shroom, a key player in apical cell constriction in neurulation, binds to ROCK2 and regulates its distribution to apical cell junctions to achieve a localized activation.…”