The G1 Cyclin-dependent Kinase CRK1 in Trypanosoma brucei Regulates Anterograde Protein Transport by Phosphorylating the COPII Subunit Sec31

Abstract

Transport of secretory proteins from the endoplasmic reticulum to the Golgi is mediated by the coat protein II (COPII) complex comprising a Sec23-Sec24 heterodimer and a Sec13-Sec31 heterotetramer. The mechanisms underlying COPII-mediated protein trafficking have been well defined, but the extent of regulation of this secretory machinery by cellular signaling pathways remains poorly understood. Here, we report that CRK1, a G 1 cyclin-dependent kinase in Trypanosoma brucei, regulates anterograde protein trafficking by phosphorylating Sec31. Depletion of CRK1 abolished anterograde transport of the secretory protein and disrupted the localization of multiple Golgi proteins, reminiscent of Sec31 depletion. CRK1 phosphorylates Sec31 at multiple serine/threonine sites, and mutation of these phosphosites to alanine recapitulates the protein trafficking defects caused by Sec31 depletion. Mutation of these CRK1 phosphosites to aspartate restored Sec31 function. Taken together, these results uncover a novel function of CRK1 in anterograde protein trafficking and elucidate the mechanistic role of CRK1 in protein trafficking through regulation of the COPII subunit Sec31.

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids

“…In summary, we highlight that knowledge concerning the duration of cell cycle phases for these human pathogens of great medical relevance is of paramount importance, not only as basic information for the development of other studies but also because of some molecular mediators that act in a cell cycle-dependent manner (Hammarton 2007;Hu et al 2016;Santos et al 2001), can be potential targets for drug development and specific life cycle-dependent therapies. Besides, we strongly suggest that all studies using BrdU, not only in trypanosomatids but also in others organisms and cell types, should be reviewed with attention to ensure an accurate estimation of DNA replication monitoring, avoiding possible mistakes.…”

“…In summary, we highlight that knowledge concerning the duration of cell cycle phases for these human pathogens of great medical relevance is of paramount importance, not only as basic information for the development of other studies but also because of some molecular mediators that act in a cell cycle‐dependent manner (Hammarton ; Hu et al. ; Santos et al. ), can be potential targets for drug development and specific life cycle‐dependent therapies.…”

COPII-dependent ER export in animal cells: adaptation and control for diverse cargo

“…Other protein kinases can also modify COPII including CK2 which inhibits the association of Sec31 with the membrane [(Koreishi et al 2013), Fig. 3Cii] as does the G1 cyclin-dependent kinase CRK1, at least in trypanosomes (Hu et al 2016). Many of the other experiments highlighting roles for phosphorylation of COPII, particularly of Sec24, point to roles in autophagy.…”

The TRAPP complex mediates secretion arrest induced by stress granule assembly

“…The function of the ERES and COPII coat is known to be under CDK control. A member of the CDK family, PCTAIRE, is recruited to ERES via its interaction with Sec23 and controls ER export (Palmer et al , ), while Sec31, a component and a regulator of Sar1/COPII cycle, can be phosphorylated by CDKs (Holt et al , ; Hu et al , ). Further, the phosphorylation of Sec31 in response to growth factor and mitogenic signals has also been reported (Olsen et al , ; Dephoure et al , ).…”

“…****P < 0.0001, Student's unpaired two-tailed t-test. ERES via its interaction with Sec23 and controls ER export (Palmer et al, 2005), while Sec31, a component and a regulator of Sar1/ COPII cycle, can be phosphorylated by CDKs (Holt et al, 2009;Hu et al, 2016). Further, the phosphorylation of Sec31 in response to growth factor and mitogenic signals has also been reported Dephoure et al, 2008).…”