Centrosomal P4.1-associated protein (CPAP) positively regulates endocytic vesicular transport and lysosome targeting of EGFR

Radhika Gudi,Viswanathan Palanisamy,Chenthamarakshan Vasu

doi:10.1038/s41598-021-91818-8

Radhika Gudi, Viswanathan Palanisamy + Show 1 more

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https://doi.org/10.1038/s41598-021-91818-8

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Abstract

Centrosomal P4.1-associated protein (CPAP) plays a critical role in restricting the centriole length in human cells. Here, we report a novel, positive regulatory influence for CPAP on endocytic vesicular transport (EVT) and lysosome targeting of internalized-cell surface receptor EGFR. We observed that higher CPAP levels cause an increase in the abundance of multi-vesicular body (MVB) and EGFR is detectable in CPAP-overexpression induced puncta. The surface and cellular levels of EGFR are higher under CPAP deficiency and lower under CPAP overexpression. While ligand-engagement induced internalization or routing of EGFR into early endosomes is not influenced by cellular levels of CPAP, we found that targeting of ligand-activated, internalized EGFR to lysosome is impacted by CPAP levels. Transport of ligand-bound EGFR from early endosome to late endosome/MVB and lysosome is diminished in CPAP-depleted cells. Moreover, CPAP depleted cells appear to show a diminished ability to form MVB structures upon EGFR activation. These observations suggest a positive regulatory effect of CPAP on EVT of ligand-bound EGFR-like cell surface receptors to MVB and lysosome. Overall, identification of a non-centriolar function of CPAP in endocytic trafficking provides new insights in understanding the non-canonical cellular functions of CPAP.

Highlights

Centrosomal P4.1-associated protein (CPAP) plays a critical role in restricting the centriole length in human cells
During our studies to determine the interaction between CPAP and other centriolar proteins, we observed that both HEK293T and HeLa cells transfected with GFP-CPAP or myc-CPAP, and confirmed the expression by immune blot (IB) (Supplemental Fig. 1A–C), formed punctate vesicular structures spontaneously
These punctate structures were observed in most cells that were transiently transfected with GFP-CPAP (Fig. 1A), and in cells that were stably expressing GFP-CPAP under a doxycycline-inducible promoter upon doxy treatment (Fig. 1B)

Summary

Introduction

Centrosomal P4.1-associated protein (CPAP) plays a critical role in restricting the centriole length in human cells. We report a novel, positive regulatory influence for CPAP on endocytic vesicular transport (EVT) and lysosome targeting of internalized-cell surface receptor EGFR. CPAP depleted cells appear to show a diminished ability to form MVB structures upon EGFR activation These observations suggest a positive regulatory effect of CPAP on EVT of ligand-bound EGFR-like cell surface receptors to MVB and lysosome. We show a novel positive regulatory influence of CPAP (centrosomal P4.1 associated protein; expressed by CENPJ gene)/SAS-4, a microtubule/tubulin binding essential centriole biogenesis p rotein[38] on EVT of internalized epidermal growth factor receptor (EGFR). By employing gain- and loss-of-function approaches as well as the ligand-bound EGFR intracellular-trafficking model, we assessed the role of CPAP in EVT and lysosome targeting of internalized cell surface receptor cargo. While the involvement of centrioles and interaction with cytoplasmic microtubules on this function of CPAP needs to be studied, our findings add a new dimension to the potential non-canonical cellular functions of a critical centriole biogenesis protein CPAP

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