Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

Abstract

CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

Diversity and plasticity in signaling pathways that regulate smooth muscle responsiveness: Paradigms and paradoxes for the myosin phosphatase, the master regulator of smooth muscle contraction

“…An active nuclear localization signal also exists in MYPT1, although PP1 binding inactivates it and interferes with MLCP nuclear accumulation (104, 105). CPI-17 gene silencing resulted in elevations in phosphorylation of histone(s) and MEF2C, but not the substrates of MLCP (103, 106). Clearly, nuclear CPI-17 inhibits a group of PP1 responsible for histone phosphorylation, but not MLCP, and likely regulates chromatin functions (Fig.…”

“…Left: haematoxylin and eosin stain; middle: anti-CPI-17 stain; right: overlay of anti-CPI-17 stain and Hoechst stain. Arrowheads indicate nuclear accumulation of CPI-17.) (103). CPI-17 consistently concentrates in nuclei of the smooth muscle cell culture under growth conditions (103).…”

“…Arrowheads indicate nuclear accumulation of CPI-17.) (103). CPI-17 consistently concentrates in nuclei of the smooth muscle cell culture under growth conditions (103). In addition, CPI-17 is expressed in a subset of cancer cells, and accumulates into the nucleus (103).…”

“…An unconventional nuclear localization signal exists at the N-terminal tail of CPI-17 (103). Ser12 within the domain can be phosphorylated (63).…”

“…Ser12 within the domain can be phosphorylated (63). Phospho-mimetic Asp-substitution at Ser12 interferes the nuclear accumulation of CPI-17 (103). Consistently, immunostaining revealed a mild exclusion of phospho-Ser12-CPI-17 out of the nucleus, suggesting that Ser12 phosphorylation plays a role in the subcellular distribution of CPI-17 (103).…”