TPPP acts downstream of RhoA-ROCK-LIMK2 to regulate astral microtubule organization and spindle orientation

Abstract

SummaryThe actin cytoskeleton in eukaryotic cells undergoes drastic rearrangement during mitosis. The changes to the actin cytoskeleton are most obvious in the adherent cells, where the actin stress fibres are disassembled, and the cortical actin network becomes more prominent with concomitant increase in cell rigidity as cells round up and enter mitosis. Although the regulatory connections between the actin cytoskeleton and the early mitotic events are apparent, the mechanisms that govern these links are not well understood. Here, we report that LIMK1 and LIMK2, the downstream effectors of RhoA and ROCK, regulate centrosome integrity and astral microtubule organization, respectively. Surprisingly, LIMK1 and cofilin are not involved downstream of RhoA and ROCK in the regulation of astral microtubule organization. Instead, we find that LIMK2 acts through TPPP in the regulation of astral microtubule organization, whereas both LIMK1 and LIMK2 affect centrosome focusing. Both phenotypes are tightly coupled to spindle orientation in the mitotic cells. Thus, our results reveal a new regulatory link between the actin cytoskeleton and the mitotic spindle during the early stages of mitosis.

Rho-associated Coiled-coil Kinase (ROCK) Protein Controls Microtubule Dynamics in a Novel Signaling Pathway That Regulates Cell Migration

“…We show that TPPP1 phosphorylation by ROCK has no effect on its tubulin polymerizing activity, suggesting that the conformational changes induced by ROCK phosphorylation do not alter its interaction with MTs. We also clearly demonstrate that ROCK1, and not the LIMKs, is responsible for TPPP1 phosphorylation, as claimed previously (24,26). As the ROCKs strongly interact and copurify with the LIMKs, the previously published findings are likely to reflect TPPP1 phosphorylation by ROCK but not by LIMKs, as demonstrated in this study.…”

“…Lysates were analyzed for LIMK1, LIMK2, GAPDH (loading control), and p-cofilin as a measure of LIMK activity. 22j (30), as it was suggested that the LIMKs phosphorylate TPPP1 (24,26)). We showed that although inhibition of LIMK activity decreased the phosphorylation of its substrate cofilin (Fig.…”

“…TPPP1 regulates MT acetylation via binding to HDAC6 and inhibiting its activity (25). Recent reports suggest that in vitro TPPP1 activity is regulated by the ROCK substrates LIM kinase 1 and 2 (LIMK1 and 2) (24,26).…”

LIMK1/TPPP1/HDAC6 Is a Dual Actin and Microtubule Regulatory Complex That Promotes Drug Resistance

“…Previous studies reported that the LIM-kinase (LIMK) family members LIMK1 and LIMK2 interact with the microtubule regulatory protein Tubulin Polymerization Promoting Protein 1 (TPPP1) [6] [8] [17]. Contrary to the initial data indicating that TPPP1 is an in vitro LIMK substrate [6] [17], we established that the LIMKs do not phosphorylate TPPP1 and that it is a substrate of ROCK in cells [9].…”

Reduced LIMK2 expression in colorectal cancer reflects its role in limiting stem cell proliferation

“…However, by focusing on how LIM kinase activity influences processes involved in cell motility and invasion, reports of negative roles of LIM kinase on functions such as cell cycle regulation may have been overlooked. Elevated LIMK2 levels following DNA damage were found to promote G 2 /M arrest via cofilin,46 which is consistent with a proposed role for LIMK2 during mitosis in regulating microtubule organisation and spindle orientation 47 48. Given that expression of the LIMK2a and LIMK2b variants has also been reported to be reduced in thyroid cancers,46 it will be important to re-consider how LIMK2 contributes to specific cancers rather than attempting to construct a unified theory.…”