# Protein Kinase D2 Regulates GRASP65 Phosphorylation and Golgi Ribbon Unlinking During G2/M Transition

## TL;DR

This study shows that PKD2 regulates GRASP65 phosphorylation, which is essential for Golgi ribbon unlinking during the G2/M transition in the cell cycle.

## Contribution

The study identifies PKD2 as a novel upstream regulator of GRASP65 phosphorylation and introduces a new phospho-specific antibody for studying Golgi dynamics.

## Key findings

- PKD2 activity is required for JNK2-dependent phosphorylation of GRASP65 at S274 during G2.
- PKD2 inhibition reduces GRASP65 phosphorylation and Golgi ribbon unlinking, delaying G2/M progression.
- A PKD2–JNK2–GRASP65 signaling axis controls Golgi disassembly at the G2/M transition.

## Abstract

What are the main findings?
PKD2 is an upstream regulator of JNK2-dependent GRASP65 phosphorylation at serine 274 during the G2 phase.PKD2 activity is required for Golgi ribbon unlinking and proper G2/M progression.

PKD2 is an upstream regulator of JNK2-dependent GRASP65 phosphorylation at serine 274 during the G2 phase.

PKD2 activity is required for Golgi ribbon unlinking and proper G2/M progression.

What are the implications of the main findings?
A PKD2–JNK2–GRASP65 signaling axis links Golgi remodeling to mitotic entry.The newly developed phospho-specific GRASP65 antibody enables investigation of upstream Golgi regulatory pathways in cell cycle control and disease.

A PKD2–JNK2–GRASP65 signaling axis links Golgi remodeling to mitotic entry.

The newly developed phospho-specific GRASP65 antibody enables investigation of upstream Golgi regulatory pathways in cell cycle control and disease.

The Golgi complex undergoes dynamic remodeling during the cell cycle, as ribbon unlinking in G2 is required for proper mitotic progression. Failure to fragment the ribbon leads to G2 arrest, whereas forced mitotic entry with intact ribbons results in multipolar spindle formation. Phosphorylation of the Golgi matrix protein GRASP65 at serine 277 (S277) in rat (S274 in human) by JNK2 is essential for ribbon unlinking, but its upstream regulation has remained unclear. Here, we generated and validated a phospho-specific antibody recognizing human GRASP65 phosphorylated at S274, enabling accurate detection of this modification. Using this tool, we identify protein kinase D2 (PKD2) as a critical upstream regulator required for GRASP65 phosphorylation and Golgi unlinking. GRASP65-S274 phosphorylation increases during G2 and is markedly reduced upon PKD2 inhibition or depletion, resulting in decreased Golgi unlinking and delayed G2/M transition. Conversely, PKD2-activating stimuli, including phorbol esters and nocodazole, enhance GRASP65 phosphorylation in a PKD2-dependent manner. These findings define PKD2 as a key regulator of the JNK2–GRASP65 signaling axis controlling Golgi disassembly at the G2/M transition. Moreover, the phospho-specific GRASP65 antibody described here provides a valuable tool to dissect upstream signaling mechanisms and to identify the initial triggers driving Golgi unlinking at G2 entry.